Sharp Honig 1990

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Annu. Rev. Biophys. Biophys. Chern. 1990. 19:301-32 Quick links to online content
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ELECTROSTATIC
INTERACTIONS IN
MACROMOLECULES:
Annu. Rev. Biophys. Biophys. Chem. 1990.19:301-332. Downloaded from www.annualreviews.org
Theory and Applications
Kim A. Sharp and Barry Honig

by Boston University on 09/12/11. For personal use only.
Department of Biochemistry and Molecular Biophysics,

Columbia University, 630 W. 1 68th Street, New York, NY 1 0032
KEY WORDS: finite difference Poisson-Boltzmann, macromolecular theory,

protein structure, DNA structure.
CONTENTS
PERSPECTIVE AND OVERVIEW.......................................................................................... 301
DIELECTRIC THEORY .... . .... . ...... . .. . .... . ... . .. ............. ....... ................ ... .. . ... ......... ............ . .... 303
The Poisson Equation ... . ...... ................... . ...... .. . .. .... . . . . .. ..... . ... . . ...... . ....... . . . ... .... . . . ... . .. 303
Electronic Polarizability ........................................................................................... 304
Permanent Dipoles .... . .. . . .. . . . ....... . . ..... ....... . . .. . .. . . . . . ............... .... ...... . ... . .. . . . .. .. ............. 307
Mobile Ions ... ...... . . ................. . ....... . . .. ....... . ................... ......... ... ... . . . . . . ..... . ...... . ... .. . .. . 309
METHODS....................................................................................................................... 310
The Protein Dipole Langevin Dipole Method............................................................ 312
Analytical Solutions t o the Poisson-Boltzmann Equation ...... . ............. ......... .......... ... 312
Numerical Solutions to the Poisson-Boltzmann Equation .......... . ............................... 3 13
The Finite Difference Poisson-Boltzmann Method ........ ......... .... . ..... . ..... . ... ........ .. . . . .. 3 14
APPLICATIONS ............................................ ................................................................... 317
Electrical Potentials around Macromolecules............................................................ 317
Solvation Energies .. . .......... . . .... . . ......... . . . ...... ...... . . ... .. . . . . .......................... . ................ 319
321
.
Catalysis .. ... ..... .. . . . . . ... .. . ... . ... . ..... .. . .. .............. .... . . ............ .. . .. ..... .. ... .... . ....................
.
Acid-Base Equilibria................................................................................................. 322

Redox Potentials ........... . . . . .. .... . .... . . . . .. .......... ........ ........ . ...... .. ........ . . . ...... . .... .... . . .. ... . . 324
Forces in DynamiCS Simulations ........ .. ................... .. .......... .. . .. . . . ....................... . . . .. . . 326
Electrostatic Energies and Stability: Folding, Denaturation, and Binding .. .... . . ..... .... 327
SUMMARY AND PROSPECTS .. .... ................ . . . . . . . . .. . . . . . .. ..... . ........ ..... .... ................. .. . . ...... .
. . . 329
PERSPECTIVE AND OVERVIEW
A recent Annual Review describes a performance at the court of Louis XV

in which an electric shock was administered simultaneously to 700 monks
301
0883-9 1 82/90/061 0-0301$02.00
302 SHARP & HONIG
who then jumped in unison to the great amusement of the onlookers (67a) .
The point of the story, in the context of the article, was that "electrostatics
was not always unfashionable." Given the recent spate of reviews on
electrostatics in biological systems (39, 47, 63, 64, 67, 9 1 , 94, 1 30), one
might argue instead that the subject has again become fashionable to the
point of overexposure. What then justifies another review?
Our major motivation has been to describe recent advances in applying
the equations and concepts of classical electrostatics to current research
areas in molecular biophysics. The fundamentals of classical electrostatics ,
like those of thermodynamics, are well known and can be stated concisely
with a few elegant equations. The apparent simplicity of these equations,
however, can hide the difficulties of applying them to complex systems.
The problem is particularly acute in studies of proteins and nucleic acids
owing to the vast amount of structural information about these macro

molecules now available. In contrast to traditional models in which pro
teins were treated as low dielectric spheres and DNA as a charged cylinder,
most current questions of interest are asked at the atomic level. The
question of how best to represent atomic and molecular properties within
the framework of electrostatic theory poses new conceptual as well as
numerical difficulties.
It is not uncommon to encounter the opinion that models based on
classical electrostatics have been superseded, or even invalidated, by the
advent of computer simulations of atomic motions. A criticism sometimes
expressed is that classical electrostatics is not valid on a microscopic scale.
In fact, excluding quantum mechanical phenomena classical electrostatics
,
is valid on all scales of biological interest. Of course, the theory must be

applied in a physically meaningful way to the system being studied. We will
demonstrate that this is generally possible and that classical electrostatics
provides a rigorous and intuitively satisfying approach to a wide range of
microscopic phenomena.
The first section gives an overview of the relevant electrostatic theory
and discusses the physical origin of each contribution to the dielectric
response of solutions of biological macromolecules. We describe the most
common representations of the relevant physical processes emphasizing
the similarities and differences between them. The second section dis
cusses specific theoretical and computational methods that have been de
veloped to calculate the electrostatic properties of macromolecular systems.
The last section reviews a range of biological problems in which electro
statics plays a central role. The framework developed in the earlier sec
tions is used to identify the relevant electrostatic quantities for a given bio
physical phenomenon and then to outline how these parameters may be
calculated.
ELECTROSTATICS IN MACROMOLECULES 303
DIELECTRIC THEORY
The Poisson Equation

The fundamental equation of electrostatics is the Poisson equation:
V2¢(r) = -4np(r), 1.
which relates spatial variation of the potential ¢ r

with position to the
charge density distribution p, where the permittivity of free space is unity.
When the charge distribution can be described in terms of a set of point

charges, the solution to the Poisson equation becomes Coulomb's law:
¢(r) = � Ir-r qj 2.
jl'
where rj is the position, and q the magnitude of the ith point charge.
j
Essentially all electrostatic models used in studying macromolecules are

based on the Poisson equation. Models begin to diverge from one another
when choices are made as to the method of applying Equation I to a
particular problem. If all charges, whatever their origin, are represented
explicitly, all interactions can be viewed as taking place in free space and
Coulomb's law can be rigorously applied. If this is not practical, or if the
details of specific i nteractions are not of interest, the Poisson equation can
be rewritten in more convenient forms. More specifically, if the charge
distribution that generates the potential is present in a complex medium,
it is possible at times to use spatial averages to account for the response
of the medium to the fields generated by the charge distribution.
If a region of some material responds in an average, or smeared out,
way to the electric field, i .e. it has a uniform susceptibility, X, the polar
ization density (induced dipole moment/unit volume) is given by: P XE, =
where E is the average field in that region. If the entire medium has a
uniform susceptibility, thc potentials and fields are screened by a constant
factor, the dielectric constant or permittivity: E 4nX + 1 . In this case, the
=
Poisson and Coulomb equations can be written in the forms

-4np(r)
V2¢(r) =
E
3.
and
¢(r) qj
I-I -I'
4.
=
j E r-rj
respectively. If the dielectric varies through space, then Coulomb's law is

invalid, while the Poisson equation becomes
304 SHARP & HONIG
V' c(r)V¢(r) = -4np(r), 5.

where c is now a function of the position r.
Generally, the Poisson equation can be applied in many ways to a
particular problem. However, unless a full quantum mechanical treatment
is used, there is essentially no way of avoiding averaging an environmental
response over some region of space (which might correspond, for example,
to a single atom or to an entire protein) . Indeed, Equation 5 says nothing
about whether a particular treatment is macroscopic or microscopic. The
definition of these terms depends solely on the spatial resolution at which

the dielectric response is modelled.
The environmental response can broadly be divided into three physical
processes that screen the effects of charge: (a) electronic polarization;
(b) reorientation of permanent dipoles, i.e. in polar materials; and (c)
redistribution of charges, such as the rearrangement of mobile ions in ionic

solutions to form electric double layers.
Electronic Polarizability
Electronic polarizability describes the reorientation of the electronic cloud
around a nucleus in the presence of an electric field. Until recently, elec
tronic polarizability has usually been neglected in potential energy force
fields used in molecular mechanics simulations because the effects cannot
be easily reduced to a set of two-body interactions. For example, if a
charge on a particular atom polarizes the electrons on neighboring atoms,
those electron clouds will also polarize one another, leading to a complex
many-body interaction.
There are a number of ways to account for electronic polarizability. The
next sections describes three of these.
THE UNIFORM DIELECTRIC MODEL When matter is exposed to radiation of
high enough frequency that nuclear reorientation cannot follow the elec
tric field, the dielectric response is determined almost entirely by electronic
polarization. The high frequency dielectric constant Ceo approximately
equals the square of the index of refraction, which is 2 for most polar and
nonpolar organic liquids. One way to account for electronic polarizability
is to incorporate its effects into a dielectric constant-to assume that all
charges and permanent dipoles interact with one another as if they were
embedded in a medium that has a dielectric constant of 2. Classical theories
of the dielectric constant of polar liquids routinely make this assumption,
while treating permanent dipoles explicitly through Equation 3 or 4 (24,
82).
A central question concerning the use of a dielectric constant over a
region of space involving many atoms asks if using a single, spatially
invariant parameter that ignores the atomic nature of matter is valid. This
problem will be considered after two microscopic models are presented.
INDUCED DIPOLES The most common means of representing electronic
polarizability at the molecular level assigns point inducible dipoles (PIDs)
to atoms, bonds, or groups (2, 1 4, 1 28) . In the simplest case, the induced
dipole moment P is presumed to be linearly related to the field by an
isotropic polarizability cc P etE. The fields arising from a charge or a
=
dipole are
E(q) = qr/r3 and E(p) = :!Tp, 6.

respectively, where :!T (3rr r2 J)/r5 is the dipole field tensor. For a
= -
collection of charges and PIDs, the field depends on the charges and dipole
moments, while each induced dipole moment in turn depends on the field
it experiences from the charges and all other dipoles. This leads to a set of
simultaneous linear implicit equations for the dipole moments:
Pi = (XiL [E(q)ij+:!TijPj), 7.
Hi
where the subscripts i and j run over all the charges/dipoles. This matrix
equation can be solved analytically only for the two-body case because, as
pointed out above, electronic polarization involves a many-body inter
action that cannot be decomposed into a sum of pairwise interactions.
Generally, an iterative procedure is used in which an initial estimate for
the dipole moments is substituted into the right side of Equation 7, and
the left side yields an improved estimate of the dipole moments. This
procedure is repeated until a self-consistent set of fields and dipole
moments results (2, 1 28) .
The PID model usually assumes that an atom has a uniform polar
izability that can be represented by an induced dipole placed at the nucleus.
Two difficulties with this model are: (a) atomic polarizabilities taken from
experiment or theory on isolated atoms are not necessarily accurate for
atoms in molecules (7); and (b) nearby inducible dipoles can mutually
increase each other's polarization without limit causing a polarization
catastrophe. The ad-hoc exclusion of interactions between neighboring
atoms has been used to circumvent this problem (97, 1 2 1 , 1 30) .
The relationship between the atomic polarizability and the bulk dielec
tric behavior of a material has been the subject of many theoretical studies,
which date back over a century to the Clausius-Mossotti relationship for
nonpolar materials:
Net
8.
x
=
(I -4nNct/3),
306 SHARP & HONIG
where N is the number density of the polarizable bodies. Although this

expression was originally derived for material with low values of N, such
as dilute gases, it also holds for denser nonpolar material (2, 14, 1 30) .
LOCAL DIELECTRIC CONSTANTS An alternate way of representing the
electronic polarizability treats atoms or groups of atoms as polarizable
bodies, each with its own local dielectric constant (LDC) (83a, 83b, 1 04,
K. Sharp & B. Honig, unpublished work) . Figure I schematically illustrates
the relationship between the uniform dielectri c, LDC, and PID rep
resentations. The LDC model effectively distributes the dielectric response

over the van der Waals volume occupied by the atom's electrons. This
model makes fewer approximations than the other two models, since it
assumes neither that th e response is uniform throughout space nor that
the response arises from infinitesimal dipoles. In the simplest form of the
LDC model, each atom is represented as a sphere of constant dielectric,

ej. The equivalent point polarizability in the PID model, (Xi, would be ( 1 4)
3 V(ej- l )
IlC = -..,.------c-
-:
, 9.
4n(ej+2)
a) Uniform b) Local c) Indue ible

Dielectric Dielectric Dipoles
qi-""i qje ""j

c=l
<j:i>/V (E-1)E/4lt E i (1+8mx)V i)

=
(1-4mx iVi)
=
Figure 1 Schematic diagram illustrating three different models for the molecular response
to electric fields: (a) Uniform Dielectric Constant (UDC), (b) Local Dielectric Constant
(LDC), (c) Point Inducible Dipoles (PID). For models a and b, the mean induced dipole
density per unit volume at any point Per) is given by ()l(r) > IV (e(r)-l]E(r)/4n, where
=
E(r) is the Maxwell field at that point and B(r) the dielectric constant. In the PID model, the
induced dipole moment at a point i is Pi = ((iEi, where ((i is the point polarizability. The LDC
model bridges the PID and UDC models through the relation ((i = 3 Vi(c,-1)/4n(6i+ 2),
relating a point polarizability to the local dielectric constant Gi assigned to a spherical volume
Vi around that point.
where V is the volume of the sphere. The LDC and PID models are
equivalent for two special cases: when the atom is in a homogeneous
medium or when it is exposed to a uniform field (K. Sharp and B. Honig,
unpublished results) . In general, however, the polarizability response
involves higher-order terms than dipoles, and on atomic dimensions the
errors in the PID approximation can be quite large ( 1 4, 83a). The LDC
model may also be extended to use nonuniform and anisotropic dielectric
distributions for each atom (83b) .
The LDC and PID models shown in Figure I appear microscopic, while
the uniform dielectric model appears macroscopic. As is evident from
Figure 1, however, the uniform dielectric and the LDC models differ in
the absence of cavities between the atoms in the former and the assumption
of the same dielectric constant for each atom. Since atoms in proteins and
nucleic acids are closely packed and are neither spherical nor static, it is
not unreasonable to consider them as filling space. Moreover, the high
frequency dielectric constant of organic liquids depends only weakly on
the identity of the solvent molecule. Thus, the use of a single dielectric
constant to account for the electronic polarization response of an entire
macromolecule appears to be a very reasonable approximation.
It should be emphasized that it is not clear which of the three models is
actually most appropriate for applications to biological systems. The PID
and LDC models are truly microscopic, but they are numerically complex
and the PID model in particular entails a number of questionable assump
tions. Moreover, both require knowledge of polarizabilities for a large
number of atoms in different molecules and thus involve a significant
number of parameters. On the other hand, the uniform dielectric model
uses a well-known experimental quantity, Boo for organic liquids, to account
for electronic polarizability. It should be informative to compare the pre
dictions of all three models for different test cases.
Permanent Dipoles
The dielectric properties of polar liquids arise primarily from the reori
entation of permanent dipoles in an electrical field. For a macromolecule
in aqueous solution, it is necessary to account for the permanent dipoles
of both the molecule and the solvent. As in electronic polarizability, it is
possible in principle to use one or more dielectric constants to describe the
response of both the solute and solvent. If a single dielectric constant is
used in conjunction with Coulomb's law (Equation 4), one is implicitly
assuming that solvent and macromolecule have the same dielectric
constant. Water has a dielectric constant of 80 at room temperature,
while experimental and theoretical evidence suggest that proteins have an
average dielectric response that can be approximated with a dielectric
308 SHARP & HONIG
constant of about 4 (30, 39, 74, 1 1 4) . Thus, the system cannot be viewed
as uniform and at least two dielectric constants must be used. We now
consider how the dielectric response of permanent dipoles can be modelled.
EXPLICIT ATOM REPRESENTATIONS The atoms and molecules in a system

are standardly represented in detail with potential energy functions in
which electrostatic interactions are described by Coulomb's law for charges,
or related expressions for higher multipoles. Because all interactions are
presumed to be treated explicitly, they can be assumed to occur in free

space, so that Equation 2 is valid. If the potential functions are accurate
and account for all relevant interactions, when combined with a method
to account for the dynamics of the system (i.e. Monte-Carlo or molecular
dynamics), they should reliably reproduce electrostatic phenomena.
The dielectric properties of macromolecules are difficult to determine
experimentally, so the extent to which all-atom models succeed in repro

ducing the molecules' behavior is impossible to assess at this time.
However, more is known experimentally about the dielectric properties of
water. These arise primarily from interactions between permanent dipoles,
and include hydrogen-bonding effects. The hydrogen bonds are reasonably
well represented with pairwise potential functions such as those of the
various water models currently in use [e.g. TTP4P (49a) or SPC (1 1 a) ] .
Simulations based on these models produce reasonable bulk dielectric
constants for water [50 for TIP4P, about 70 for SPC (5, 7 5)]. However,
there remain formidable problems of computational expense, potential
function parameterization, convergence behavior, cutoff, and limitations
of the current pairwise point potential formalisms (39) . The long-range
nature of electrostatic interactions compounds the difficulty. Moreover,
water models that account for electronic polarization are in their earliest
stages of development and introduce significant additional computational
requiremen ts.
The problem is exacerbated when simulating waters around macro
molecules. In this case, the size of the macromolecule limits the number
of water layers that can be included in a simulation. This limitation and
the use of boundary conditions whose effects are unclear introduce con
siderable uncertainty about the dielectric response of waters that appear
in the simulations, even if the potential functions produce reasonable
dielectric constants for bulk water.
STATISTICAL MECHANICS REPRESENTATIONS The Langevin model The

Langevin equation relates the mean orientation e of a freely rotating
dipole to the magnitude of the directing field Ed:
1 0.
This equation represents the balance between the polarizing effect of the
field and the randomizing effect of thermal fluctuations, where T is the
absolute temperature and k is Boltzmann's constant. Onsager (82) used
this model to calculate bulk dielectric behavior. In principle, the Langevin
model is applicable only to freely rotating polar molecules, i .e. those whose
only interactions are electrostatic. Moreover, because it ignores correlated
motion between solvent molecules, it cannot account for the dielectric
constant of hydrogen bonding liquids without some type of correction
( 1 4), nor does it properly represent the effect of dielectric saturation or

electrostriction around ions (48 ) . However, by suitable parameterization
it has been used to represent even a highly associated liquid like water
( 1 30) .
Kirkwood-Frohlich theory Many detailed statistical-mechanical treat
ments of orientational polarization of polar dielectrics are based on the

Kirkwood-Frohlich theory (28, 52). Here, the material is considered as a
collection of permanent dipoles embedded in a continuum with a high
frequency dielectric constant of eoo. The effective dipole moment of each
dipole thus increases from the gas phase value due to polarization by its
reaction field. Thc thcory treats a spherical region of material in molecular
detail by statistical mechanics, representing the rest of the material as a
continuum:
9kT(e-eoo)(2e+eoo) 2
= gf-l , 11.
4nNs(soo +2)2
where c is the bulk dielectric constant, N is the dipole number density, f-l
is the gas phase dipole moment, and 9 is the Kirkwood correlation factor
that accounts for cooperative effects orienting neighboring dipoles. Kirk
wood-Frohlich analysis has been used to theoretically estimate the dielec
tric constant of a protein. Assuming a hypothetical oc-helical protein of
infinite extent (to avoid boundary problems) , Gilson & Honig (30) esti
mated a value between 3 and 4. Nakamura & Nishida (73) extended this
analysis by calculating a dielectric constant as a function of position in the
protein Pancreatic Trypsin Inhibitor (PTI) . Numbers ranging between 3
and 4 were also found for the protein itself. Although Nakamura &
Nishida (73) obtained much higher effective dielectric constants when they
averaged the dielectric constant of water into the values assigned to atoms
on the protein surface, the essential conclusion in both studies is that
proteins, when viewed as pure materials, have a low dielectric constant.
Mobile Ions
THE POISSON-BOLTZMANN EQUATION In this mean-field approach, mobile
ions in ionic solvents are not represented explicitly. Instead the chemical
3lO SHARP & HONIG
potential of each ion is assumed to be uniform throughout solution. The

entropic and electrostatic contributions to the chemical potential of an ion
at any point rare kTln C(r) and q</J(r), respectively, where C(r) is the
local concentration, q is its charge, and </J(r) is the mean potential. This
leads to a Boltzmann expression for the ion concentration:
C(r) = C( (0) exp [q</J(r)lkT], 1 2.
where C( (0) is the bulk ion concentration. When incorporated into the
Poisson equation, this yields the most general of the widely used continuum
electrostatic equations, the Poisson-Boltzmann (PB) equation:
V' s(r)V</J(r)-K,2 sinh [</J(r)] = -4np(r), 13.
where the Debye-Huckel length 11k is related to the ionic strength I by:
8nNae2 I
K2 = K,2 Ie = 14.
(1000skT)'
where Na is Avogadro's number. Equation 1 3 incorporates electronic and
dipole polarization through s and ion screening through K, and it allows
shape effects to be modelled through the spatial variation of B, K, and p.
EXPLICIT ION REPRESENTATIONS It is also possible to represent mobile ion
screening around polyelectrolytes explicitly through Monte-Carlo simu
lations (57, 68, 69, 72) or integral equation methods (8), thereby including
finite size and correlation effects for the mobile ions. To date these studies
have used the primitive model in which the ions are explicitly represented,
while the water is represented as a continuum of dielectric 80. For small
univalent ions, discreteness effects appear to cancel to a large extent, and
the agreement of results using the primitive model with PB equation
solutions is good (72). Agreement is not as good for divalent cations,
although the qualitative predictions of the PB equation remain valid. Early
studies used simple representations of the polyelectrolyte and its dielectric
behavior, but more recent work includes shape effects (8) and, in com
bination with the Poisson equation, dielectric boundary effects (18, 119).
Very few simulations have been performed with explicit ions and water,
and then only with a small number of ions (1 2, 100).
METHODS
The previous section summarizes the physical interactions that underlie

the response of biological systems to electrical fields. In this section we
review the methods currently available to calculate these interactions.
Any calculation, independent of model, starts with some set of assump-
ELECTROSTATICS IN MACROMOLECULES 31 1
tions about the molecular structurc, chargc distribution, and cnviron
mental response of the system of interest. The environmental response is
described with a simulation and statistical mechanical averaging, a dielec
tric constant (which may vary throughout space), or some other rep
resentation of polarizability, such as inducible point dipoles located at
atomic nuclei. The response of mobile ions is generally represented through
the PB eq uation or with a Monte-Carlo simulation. Thus, all models can
be reduced to point (or smeared) charges and dipoles interacting with each
other and with their environment. In practice, we need to deal only with
charges, since most treatments break down dipoles into pairs of partial
charges.
For an assembly of point charges, qh the total electrostatic energy
relative to that with all charges infinitely separated in vacuum, is
15.
where <Pi is the potential experienced by charge qi ' The potentials can be
decomposed into terms arising from each charge (35), so that
1 6.
where <Pfj (x = c, e, s, or m) is the potential at charge i due to charge j. I

The potentials <Pfj describe the vacuum Coulomb interaction between all
pairs of real and partial charges in the macromolecule; <P�j describes the
interaction of the charges with the polarizable electrons in the molecule.
These are the two intramolecular interactions. The potential terms <P�j
and <PV describe the interactions with the solvent molecules and solvent
mobile ions, respectively.
With the exception of the vacuum Coulomb potential <P�, each term <P�
in Equation 1 5 contains a self-energy term from the electrical potential
induced by charge i at its own position, r/J�i' in the absence of other charges.
This energy can be thought of as a solvation energy; solvation is broadly
defined as the interaction of a charge with its own induced polarization.
The pairwise interaction energies result from the contributions to <Pi from
each other charge}. This potential has contributions from electronic polar
izability <P�j, solvent molecules <P�j, and mobile ions <P'!}, which all screen
the direct Coulomb interaction r/JYj• These screening effects, instead of
being considered explicitly, are often combined into an "effective dielectric
I The complication introduced by the nonlinearity of ¢m is ignored here.

312 SHARP & HONIG
constant." When using this kind of term in a particular context, one must
define exactly what physical interactions the term is intended to describe.
The methods that have been developed for the calculation of electrostatic
interactions differ in their treatment of the various terms in Equation 1 6.
The crucial difference is in the method used to average the response of the
environment to the charge distribution. In the following, we summarize the
most commonly used approaches and emphasize the averaging procedure
implicit in each and the assumptions used in treating the various terms in
Equation 1 6. The excellent review by Harvey (39) should be consulted for

a more extensive discussion of the topic.
The Protein Dipole Langevin Dipole Method

This approach (abbreviated PDLD here) was developed by Warshel and
coworkers (128, 1 30). The PDLD method treats electronic polarizability

by calculating all charge-induced dipole interactions according to Equa
tions 6 and 7 so that each atom is assumed to have a uniform polarizability
represented in the point dipole approximation (see above discussion of the
PID treatment of electronic polarizability) . The solvent is treated by plac
ing point dipoles on a lattice and having these dipoles respond according
to a Langevin expression similar to Equation 1 0 that contains an additional
parameter to facilitate the calculation of solvation energies. Ionic strength
effects are not treated in this model.
In the PDLD method, the solvent is assumed to have a uniform dielectric
response (polarizability), represented by discrete point polarizabilities. The
shortcomings of the PDLD method include the problems associated with
using point dipoles to represent both electronic and solvent polarizabilities
(see above) and the fact that the iteration procedure used to solve Equation
1 0 applies to the magnitude but not to the direction of the Langevin dipoles
(39). Nevertheless, the method has worked extremely well for the problems
to which it has been applied. In particular, its ability to treat the self
energy contributions to ¢e and ¢' in Equation 1 5 made it possible to treat
electrostatic contributions to enzyme catalysis in a physically rigorous
fashion for the first time.
Analytical Solutions to the Poisson-Boltzmann Equation

Calculations based on the PB equation generally assign the macromolecule
a uniform low dielectric constant, while the solvent is assumed to have the
dielectric constant of water (about 80). Equation 13 can then be solved
analytically only when simplifying assumptions are made about the shape
of the macromolecule. Also the sinh (4)) term in the PB equation must
often be linearized. Despite these sometimes drastic assumptions, sim-
plified theories based on the PB equation have been remarkably successful
in treating a wide range of phenomena.
Perhaps the greatest successes have been obtained for the electrical
potentials in the solvent near planar membranes. The charge distribution
is assumed to be smeared over the membrane surface and solutions are
easily obtained using Equation 1 3 (Gouy-Chapman theory) . This classical
theory, as well as more recent developments in the theory of membrane
electrostatics, are reviewed in McLaughlin's article (67) and are not con
sidered further here.

Solutions to Equation 1 3 for a uniformly charged cylinder have been
used to study the electrostatic properties of DNA (29, 37, 55, 1 34). Another
polyelectrolyte theory not derived directly from the PB equation is Man
ning's condensation theory (62), which predicts that a fraction of the
counterions around DNA are territorially bound, or condensed. Con

densation theory relates to the PB model in that they both balance the
en tropically favorable dispersion of counterions with their enthalpically
favorable attraction to the charges on a polyelectrolyte. In fact, Zimm &
LeBret ( 1 34) demonstrated that cylindrically symmetric PB solutions and
condensation theory give the same value for the condensed fraction.
The Tanford-Kirkwood ( 1 1 6) theory (T-K) , which has been widely
applied to proteins, treats the protein as a low dielectric sphere, with
embedded charges, surrounded by a high dielectric solvent. The linearized
form of Equation 1 3 solved for this system leads to a series solution for
the electrostatic potential. This method has been widely applied to protein
titration curves, most recently with a modified approach developed by
Gurd, Matthew, and colleagues (63, 64, 1 05) . These workers succeeded in
mapping some three-dimensional information onto the spherical model of
a protein, in part through the application of an additional screening factor
of (1- !.) to the potential from each charged atom, where!. is the fractional
accessibility of that atom. The use of this factor has been difficult to justify
on physical grounds, but it has nevertheless been effective in improving
experimental agreement for protein titration curves. The method has
additional, implicit, theoretical difficulties [see discussion by Harvey (39)],
but it nevertheless represents an important step in the development of
methods that map protein structural information onto the parameters of
the PB equation.
Numerical Solutions to the Poisson-Boltzmann Equation

The major shortcoming of the analytical methods summarized in the
previous section is the assumption of simplified shapes for the macro
molecule. While this makes it possible to study global properties, the
approximations involved are less appropriate for the examination ofprob-
314 SHARP & HONIG
lems at the level of individual residues and of phenomena that depend on

the detailed shape of the macromolecule. In recent years, numerical
methods have been developed that solve the PB equation for complex
shapes and charge distributions so that now one can treat a wide range of
phenomena without using any simplifying assumptions other than those
inherent in the PB equation itself.
Pack & Klein (54, 86) developed a method of obtaining a self-consistent
set of potentials and mobile ion charge densities using an iterative tech
nique in which Coulomb's law is used to determine the potentials and

Equation 1 2 is used to determine the charge densities. This method was
applied to a number of problems in DNA structure and made possible an
account of the shape of the macromolecule in an analysis of ionic strength
dependent phenomena. By using Coulombic potentials, however, this
method implicitly assumed a uniform dielectric constant in both solute

and solvent.
Harvey (39) provides a useful summary of the development of numerical
solutions to the PB equation. Orttung (83), who used a finite element
method, reported the first numerical solution to the Poisson equation for
a molecule in which the differing polarizabilities of solute and solvent were
explicitly taken into account. Warwicker & Watson ( 126), who used a
finite difference approach, presented the first numerical solution to the
Poisson equation for a macromolecule. Subsequently, finite difference
solutions to the linearized PB equation were reported by Warwicker ( 123)
and by Klapper et al (53) . More recently, the full nonlinear PB equation
has been solved with finite difference methods (49). At this time, a number
of groups (II, 22, 23, 77, 96) have independently written finite difference
programs, and use of this approach has become fairly widespread.
Boundary element methods have also been used to solve the Poisson
equation (90, 1 33) but have had difficulties dealing with the PB equation.
Nevertheless, these methods provide interesting alternatives to the finite
difference approach and warrant further exploration.
The Finite Diffe rence Poisson�Boltzmann Method

The finite difference Poisson-Boltzmann (FDPB) method has become
widely used in recent years. This section briefly reviews the method's
implementation.
In the FDPB method, the macromolecule(s) and a region of the sur
rounding solvent are mapped onto a cubic lattice; each lattice point rep
resents a small region of either the molecule(s) or the solvent. Values are
assigned at each point for the charge density, dielectric constant, and ionic
strength parameters in the PB equation. With a fine enough lattice scale,
variation in dielectric response can be represented at the atomic resolution.
Though applications published to date have generally averaged the dielec
tric constant over the entire molecule, the FDPB method, like the Poisson
equation itself, is neither intrinsically macroscopic nor microscopic; this
issue depends only on the assumptions used in its application.
DEFINING THE MOLECULAR SURFACE AND ATOMIC CHARGES Most studies

use the solvent accessible surface (58) to define the macromolecule-solvent
boundary, using a water probe radius (typically 1.4 .A). The radius and
charge of each atom is usually taken directly from some force field. Little
effort has gone into obtaining new parameters for FDPB calculations and
indeed, it seems most reasonable to avoid the further proliferation of
parameters. It is however, necessary to check how well a particular pa
rameter set actually works; solvation energy calculations discussed below
may provide the optimal check.
THE SOLVENT DIELECTRIC CONSTANT All lattice points within the solvent
region are assigned a solvent dielectric constant and an ionic strength
parameter. To date, the solvent has been treated as a continuum, with
water molecules seen in the x-ray structure assigned either the dielectric
constant of bulk solvent or of the macromolecule. A major concern is the
extent to which the use of a bulk dielectric constant is appropriate. There
is no difficulty within the FDPB method in assigning a reduced dielectric
constant to different solvent regions such as boundary water; the problem
is rather what value to use. For example it is not clear that the bound
waters observed in the x-ray studies are present outside of the crystal
environment. Recent NMR evidence (84) suggests that waters near pro
teins and nucleic acids are quite mobile, indicating a high dielectric
constant. A number of recent protein simulations have also found bound
ary water to have short relaxation times (1). Nevertheless, this question
obviously deserves further study.
THE INTERNAL DIELECTRIC CONSTANT The assignment of a dielectric con

stant to lattice points within the macromolecule depends both on the
physical model and on the application. Consider, for example, a problem
in which the conformation of a macromolecule is assumed to be fixed.
Since the dipolar groups are fixed (or thought of as being in a time
averaged position), they make no contribution to the dielectric constant.
Under these circumstances, one need account only for the contributions
of electronic polarizability. With a fine enough lattice, it is possible to
assign a different dielectric constant and, hence, polarizability to every
atom (e.g. using the LDC model, Equation 9). An alternate approxi
mation currently in use is to use a uniform dielectric of two, based on
316 SHARP & HONIG
the high-frequency dielectric constant of organic liquids, for the entire

macromolecule.
In many applications, the macromolecular conformation would be ex
pected to change as a result of some process (ion binding, titration, etc) .
The response of the permanent dipoles as well as electronic polarizability
must be accounted for in this case. Two strategies suggest themselves. If
the change in conformation is known from x-ray analysis or predicted
from theory, the contributions of the permanent dipoles can be treated
explicitly in each conformation and only electronic polarizability need be

incorporated in the dielectric constant. If the change in conformation is
not known, the effects of permanent dipoles can be incorporated in an
average way into the dielectric constant by using a value greater than two.
As discussed above, theoretical and experimental arguments suggest a
value of 3-4. A value of 4 has been used in most applications, which seems
to work quite well for the problems we have studied. It should be stressed
that in cases where regions of the protein are expected to significantly
rearrange, for example, upon ion binding, using a dielectric constant to
describe the response of the atoms in question would be inappropriate.
NUMERICAL CONSIDERATIONS The choice of lattice size is a trade-off

between higher resolution of a finer grid versus the increased memory and
time requirements. One solution, which also increases numerical accuracy,
is to use focussing, which makes it possible to progressively increase
resolution using boundary conditions from a previous run (36). The recent
development of more rapidly converging algorithms for the linear FDPB
method makes larger lattice sizes more practical (23, A. Nicholls & B.
Honig, unpublished results) . For example using the algorithm recently
developed in our lab, a single calculation with a lattice order of 65 (a
reasonable choice) takes only about 30 seconds of CPU time on a Convex
Cl.
Values for the potential must be estimated at the edge of the lattice. The
lattice spacing must be chosen so that the minimum distance between the
molecular surface and the lattice boundary is large enough for accurate
analytical approximation (obtained, for example, using Debye-Huckel
potentials) (36) . In practice we have found that setting the longest dimen
sion of the molecule to be approximately two-thirds of the lattice dimension
is a good compromise. If necessary, improved accuracy can be obtained
by focussing or using the variable boundary method (74) to determine the
final boundary values. Periodic boundary conditions (49) allow infinite
spans of repeating units of DNA or membrane to be represented.
Finally, it is important to estimate the precision by comparison with
suitable analytical solutions, to establish that convergence has been
reached, and to check the sensitivity of the results to all the parameters
used to treat a particular problem. Variations in potential may arise due
to the way the system is mapped onto the lattice, but either rotational or
translational averaging, which displaces the molecule on the lattice, can
be used to check for and reduce any errors arising from this source (36).
In applications of the FDPB method reported to date, numerical errors
do not appear to be a limiting factor.
DYNAMICAL EFFECTS The FDPB method calculates the electrical potential

for a static structure. Small fluctuations can be incorporated into the
dielectric constant as discussed above, but cases may arise in which the
static picture is either incorrect or misleading. In characterizing electrical
potentials around macromolecules or the interaction energy between two
sites on a protein, it may be necessary to consider whether conformational

fluctuations will alter the picture provided by a static, time-averaged struc
ture. A recent molecular dynamics study found that significant fluctuations
in electrical potential occurred during the course of the simulation ( 1 32) .
Determining, through statistical mechanical averaging, whether these
fluctuations have a significant effect on different physical observables
would prove interesting. On the other hand, the most meaningful test of
this question is the extent to which it is possible to use a static model to
reproduce experimental observations on different systems. The success of
the FDPB and PDLD methods in accounting for a wide range of phenom
ena (see next section) suggests that in many (although certainly not all)
applications, analysis based on static or time-averaged structures suffices.
APPLICATIONS
Electrical Potentials around Macromolecules

Charged residues on the surface of macromolecules can generate electrical
potentials that extend many angstroms out into solution. In DNA, the
potential produces a large concentration of closely associated counter
ions, which is a major factor in conformational and binding properties
(6) . For a discussion of these ion distributions and their relation to PB
equations, see the chapter by Anderson & Record in this volume (6a) .
The electrical potential around proteins can enhance substrate association
rates, and the electrical potential around DNA is believed responsible
for non-sequence-specific binding of proteins and other molecules. The
electrical potential around certain proteins may also be responsible for
DNA bending (8 1 , 1 24, 1 25) .
318 SHARP & HONIG
The FDPB method provides the most accurate way available of cal
culating a potential around a macromolecule. Detailed simulations do not
extend far enough from the surface to be meaningful, nor do they inelude
ionic strength effects. The PDLD method is also not appropriate for
calculating these potentials. In some cases it is not unreasonable to use
Coulomb's law with a dielectric of 80 or use analytical solutions based on
simplified models of the macromolecule (4 1 ) . However, in general the
shape of the dielectric boundary between macromolecule and solvent can
have important effects on electrical potentials that will be lost if simplified

methods are used.
The most dramatic shape effects appear due to the focussing of electric
fields by clefts or channels. These structures can induce large potential
gradients within active sites (22) or magnify the potential out into solution.
Potentials generated in the solvent have been shown to have large effects
on the association rates of charged molecules. This phenomenon has

recently been studied extensively with Brownian dynamics calculations,
which make possible simulation of the diffusion process (27, 56, 78) . The
method that has been most effective for studying association uses potentials
generated from FDPB calculations as a basis for calculating the forces
used in the Brownian dynamics simulations (4, 1 0 1 , 1 02). Applications to
the protein Cu, Zn-superoxide dismutase successfully accounted for the
observed (20) magnitude and ionic strength dependence of the rate con
stant for superoxide diffusion to the active site (3, 1 0 1 ) . Models based on
simple Coulombic potentials gave results that conflicted with experimental
data ( 1 02) .
Recent developments by Northrup and co-workers (78) now make it
possible to treat rotational as well as translational diffusion. This is essen
tial in problems involving the association of two macromolecules. For
cytochrome c-{;ytochrome c peroxidase association, the electrical potential
appears to affect both translational and rotational diffusion, ensuring that
the two redox partners are properly aligned for electron trapsfer.
Calculations on nucleic acids have also revealed interesting shape effects.
Ion concentrations around DNA are found to be enhanced above the
prediction of Coulombic models (49) . In tRNA, a remarkable "window"
in the negative potential around the molecule is found in the anticodon
region ( 1 03). This serves to reduce the repulsive interactions with other
negatively charged molecules and may thus enhance specific interactions
with mRNA.
To calculate potentials around a molecule, it is only necessary to define
the van der Waals surface and charge distribution. A single run of a FDPB
program will produce potentials on a cubic lattice that can be displayed
graphically or read out at specific locations.
Solvation Energies
The calculation of aqueous solvation energies is centrally important
because molecules necessarily change their solvation state in all processes
involving binding or conformational rearrangements. For example, when
a substrate binds to a protein, the interaction of both molecules with the
solvent changes as a result of the binding process. We discuss binding and
conformational energies in a later section; here we consider the process in
which a molecule is transferred from one medium to another. In particular,
considerable experimental and theoretical effort has been invested in recent

years in studying the energetics of transferring small solute molecules from
the gas phase to water.
The gas phase is a convenient reference state for studying solvation
because in this state no solute-solvent interaction energies are present.
Thus, the energetics of transfer depend only on solute-water interactions.
The solvation process involves cavity formation, changes in van der Waals
interactions, and electrostatic factors. In this section we discuss only elec
trostatic contributions to solvation. Electrostatic interactions are the domi
nant factor in the solvation of charged groups, however, and are generally
the largest single term for dipolar groups as well.
Each charge in a molecule creates a field to which the environment
responds by electronic polarization and rearrangement of charge, which
results in a reaction potential back at the charge (p;. From Equation 1 6,
the energy of a collection of charges qi in this reaction potential, flG" can
be written:
flGf = L 3 (¢�+¢;+¢';') = L qi¢�!2. 1 7.

I I
The change in flGf upon transfer of a charged or polar solute from one
solvent to another defines the electrostatic component of the solvation
energy. (pr contains contributions from the solvent cjJs and mobile ions cjJffi,
if they are present.
Solvation energies can be calculated in a number of ways. Free energy
perturbation methods have been used extensively in recent years to obtain
vacuum-to-water solvation energies [(9, 1 6, 60) see Beveridge and co
workers (12, 68) for a review] with generally good results. The PDLD
method also yields good agreement with experiment ( 1 30). The Poisson
equation serves as a basis for obtaining electrostatic contributions to
solvation energies in a third option.
The simplest example of the successful application of the Poisson equa
tion is the Born model for ion solvation. Although the ion was originally
treated as a uniformly charged sphere ( 1 3) , the ion can also be treated as
a point charge in a sphere if one follows the formalism of Equation 1 7.
320 SHARP & HONIG
The change in solvation free energy .1 .1 G solv in transferring an ion of radius

a and charge q from a medium of dielectric constant el to a medium of e 2
is:
.1.1G solv q .1 tjJr q 2 ( 1 /82 - 1/8 1 )

= =
1 8.
2 2a '
The reaction potential formalism opens up the possibility of using the
Poisson equation to obtain solvation energies for more complex systems
(32, 90) .
Rashin & Honig (89) showed that the Born radius can be identified
with the cavity radius formed by the ion. Use of this radius reproduces
experimental transfer energies (to within 1 0%) for ions ranging in charge
from - 1 to + 4. Recent integral equation calculations show that it may
be possible to uniquely define a Born radius in tcrms of the paramcters
standardly used in intermolecular potential functions (44, M. Karplus,

personal communication) .
The success of the Born model is perplexing because it ignores the
structural properties of the solvent molecules. However, free energy simu
lations have confirmed one of the key assumptions of the model, that
dielectric saturation near the ion is not an important factor, partly because
of the opposing effects of electrostriction (48) . The success of the Born
model in reproducing solvation free energies of small ions strongly encour
ages the use of the Poisson equation in other solvation calculations.
In this regard, the Born model currently gives better results for solvation
energies of ions than do free energy simulations, which tend to overestimate
solvation energies, particularly of cations ( 1 6, 48, 1 1 3) . This is believed to
be due in large part to the neglect of three-body electronic polarization
interactions, leading to underestimation of the mutual repulsion of the
first shell waters. This discrepancy should reduce as polarization terms
are included in potential functions but only at increased computational
cost.
As pointed out above, the calculation of solvation energies of molecules
treated as point charges inside a van der Waals envelope involves cal
culating the reaction field at the positions of each of those charges. In any
determination of reaction fields, one calculates the potential induced by a
charge at the location of that charge. To do this it is necessary to subtract
out the direct potential of the charge on itself (i.e. renormalize) . In force
field and PDLD calculations, one simply omits this potential from the
summation of pairwise potentials. For numerical solutions of the PB
equation, it was recently pointed out that this term is automatically sub
tracted out when calculating differences in potential in different solvent
environments (32, 90) . Once this is realized, obtaining electrostatic con-
tributions to solvation energies, binding energies, pK values, redox poten
tials, etc is straightforward.
A number of solvation energies calculations using the Poisson equation
have been reported. Rashin and Namboodiri (88, 90) used boundary
element solutions to obtain electrostatic contributions to solvation
enthalpies. They added empirical estimates of the cavity terms, hydro
phobic energies, and van der Waals contributions to achieve solvation
enthalpies in good agreement with experiment. The FDPB method has
been used to obtain solvation free energies of charged molecules also in

good agreement with experiment (32). In a collaborative effort with Clark
Still's group at Columbia, we have used the free energy perturbation,
PDLD, and FDPB methods to calculate electrostatic contributions to
solvation free energies for 14 polar and ionic solutes, with solvation ener
gies ranging from 3.5 to - 80.0 kcal/mole. With a few exceptions all
three methods give very similar results, although the free energy per
turbation results are too negative for charged groups, probably for the
reasons given above. Rashin (88) has also found that his boundary ele
ment method gives results similar to those obtained from Monte-Carlo
simulations.
Catalysis
As discussed in the previous section, the electrical potential of charged
amino acids on the surface of proteins can influence association rates.
Another postulated role for surface charge is the enhancement of catalytic
rates in enzymes. The PDLD method has been used for some time to study
the role of charged amino acids within the active site. At present, the
FDPB method is the only one that has been used to study the effects of
surface charge. If an FDPB calculation is used, potentials in the active site
generated by one or more amino acids are calculated, and electrostatic
energies are obtained by multiplying these potentials by the appropriate
charges assumed to be present in various steps in the catalytic mechanism.
If, for example, a proton transfer is of interest, the potential difference
between the two sites, multiplied by the proton charge provides the con
tribution to the energetics of this step.
A number of FDPB calculations of the electrical potentials in the active
sites of enzymes have been reported. In a study of actinidin and papain,
electrostatic fields due to charged amino acids were evaluated and suggested
to be the basis for the reactivity characteristics of these enzymes (87). A
similar conclusion was reached for lysozyme, where a sharp potential
gradient was found across the active sites of lysozymes with dissimilar
amino acid sequences (22). It was suggested that a significant part of this
field arises from the clustering of positive charges on one lobe of the
322 SHARP & HONIG
protein and from the focussing of field lines through the active site. In
contrast, a recent study of trypsin isozymes proposes that surface charge
plays little or no role in these proteins ( l OS) . Indeed, rat and cow trypsin,
which differ in charge by 1 2. 5 units, have essentially identical active site
potentials.
For trypsin, eletrostatic stabilization of the transition state is extremely
important, hut the entire effect seems due to hydrogen bonding groups
and to the Asp- 1 02 that is part of the active site catalytic triad. Both
PDLD ( 1 29) and FDPB calculations ( 1 08) found that Asp- l 02 stabilized
the transition state by about 4-5 kcaljmole, in excellent agreement with
experiment (19). That the results of the two methods are so similar is
encouraging.
For lysozyme, Warshel & Levitt ( 1 2S) argued some time ago that the
electrical potential of Asp-52 played an important catalytic role. The
calculations of Dao-Pin et al (22) show a comparable effect from the

surface charge.
Acid-Base Equilibria
The main problem in acid-base equilibria is to determine the protonation
state of each ionizable group in a macromolecule. This state depends on
its environment, the presence of other ionizable groups, and the bulk
pH. Consider. an " ionizable group i that is part of a macromolecule. The
fractional degree of proton dissociation, ii' is given by
log [fJ( l - f;)] =
(pH - pK;) 19.
where pKi i s the p K o f the group a t that pH, given by
pK; = pK :nt + L1G jtrj(2.303kT) = pK 0 + (L1dGfnv + dGltr)/2.303kT, 20.
where pK int is the intrinsic pK of the group in the macromolecule, i .e. the
pK it would have if all other charges were absent. The dGltr term accounts
for the pK shift due tq the potential at site i arising from the other titratable
groups in the macromolecule. This term is thus a function of the fractional
dissociation state of these other groups, jj, and hence, unlike pK int, it is
pH dependent. pK int comprises two terms: pK 0, the reference state p K
(e.g. the pK of the isolated group in water, which i s usually known) and
dL1Genv, the extra electrostatic energy of dissociating the proton in its
macromolecular environment relative to its reference state.
The pK 0 in water may be calculated from the vacuum reference state
dissociation energy and the energies of interaction with the environment
dG env of HA, H + , and A - (or H+ A, A, and H+ for basic groups) (46, 50,
1 27) . In water, L1Genv for each species equals its solvation energy, L1Gso1v.
Thus the solvation energy contribution to the dissociation process in water,
IlIlG SOlv (W) , is the difference in solvation energies between HA, and H+
and A - . The corresponding contribution in the macromolecule for the
process M AH -+ M-A - + A + is the difference in the energies of interaction
-
with the environment IlGenv of M -AH, M-A - and H + , where IlGcnv for
each species contains its residual interaction with the solvent plus its
interaction with the polarizable electrons and permanent dipoles in the
macromolecule. Thus we can write:
IlIlGenv = IlIlGso1V (w) - IlIlGcnv (M)
=
[IlG so1V (A - L - IlG so1,(AH)w]
- [IlGenv(A - ) M - Il Genv (AH) M] ' 21.

In practice electrostatic energies o f the neutral species are small compared
with those of the charged species, and IlIlGenv may be obtained from the
difference between the solvation energy of A (or H + A) in water and in

-
the macromolecule.
Since the solvation energies of charged amino acids are large ( � 70
kcal/mole) yet changes in intrinsic pK values are usually small ( � 1-2 pK
units), the total electrostatic energy in the macromolecule must also be on
the order of � 70 kcal/mole. Since intrinsic pK values depend on the
differences between two large numbers, their calculation provides a par
ticularly challenging test of any theory of acid-base equilibria ( 1 30). Russell
& Warshe1 (98) have used the PDLD method to calculate the intrinsic pK
values of various acidic groups in PT!, and Warshel et al ( 1 3 1) more
recently extended this work to include explicit treatment of solvent mol
ecules. The FDPB method has been used to calculate intrinsic pK values
in lysozyme ( 1 0) and trypsin ( 1 08). Good agreement with experiment was
obtained in both cases.
If one is interested in changes in pK due to the presence of other charged
groups or resulting from alterations in ionic strength, then pKint can be
ignored and one needs to calculate only IlGltr , For example, the effect of
a single charged group, j, on the pK of another group, i [as probed for
example with site-directed mutagenesis ( 1 1 8)], is given by the potential
produced by j at the site of i, 4>ij (assuming an unchanged protonation
fraction for all other groups) , The FDPB method, applied to this type of
problem, has yielded excellent agreement with experiment for subtilisin
(3 1 , 1 1 2) and lysozyme (22) ,
To determine a protein titration curve, it is necessary to calculate sim
ultaneously the ionization state of all the ionizable groups at each pH
value, and thus the net molecular charge. Since the potential at each group
depends on the ionization state of all the other groups, which in turn
depend on the potentials at these groups, it is necessary to iterate the set
324 SHARP & HONIG
of h and cPj to a self-consistent solution ( 1 1 7) . The T-K theory, in its

modified form, has been most successful in this area. It has not been used
to calculate intrinsic pK values, but an empirical procedure of adjusting
the intrinsic pK values by ± 0.5 (64) allows one to obtain good fits to
experimental titration curves. Since only the net molecular charge is
required, the calculations are less sensitive to the uncertainty in potential
and pKj of any single group.
Bashford & Karplus ( 1 0) recently reported the first FDPB calculation
of a full titration curve. Instead of assuming intrinsic pK values, they

calculated them explicitly and obtained charge-charge interactions as a
function of pH. The results were generally in good agreement with experi
ment. The ilG :tr values in Equation 20 are functions of the mean potentials
averged over all the different protonation states of the macromolecule. In
the original procedure of Tanford & Roxby ( 1 1 7) , and other calculations
to date, the mean potential was assumed to be proportional to the

mean charge, and hence the mean occupancy, of a group, i.e.:
< cPlh¥;) oc <h)qj· This assumption neglects the configurational factors
associated with the different states of dissociation for such a multiple
site equilibrium ( 1 1 5, 1 22). Bashford & Karplus ( 1 0) found that this
approximation can lead under certain conditions to significant errors and
have developed an alternative statistical mechanical approximation.
Finally, a closely related problem, calculation of proton exchange rates
in proteins, is worth mentioning. Because the transition state in the ex
change process involves the dissociation of the proton, the pKa of the group
affects the rate. Considerable experimental data have been accumulated on
hydrogen exchange rates in proteins (e.g. 1 5) . Calculations of exchange
rates in lysozyme using a model intermediate between the T-K and FDPB
models ( 1 1 0) have been made with good results for pH dependence of the
base-catalyzed exchange rate (25) .
Redox Potentials
Electron transfer proteins usc a small number of redox cofactors to carry
out a wide range of functions. Since redox potentials vary over many
hundreds of millivolts, the electrochemistry of molecules such as hemes,
chlorophylls, flavins, and Quinones is modified by the molecules' associ
ation with proteins. This modification provides a flexible range of redox
midpoints, allowing fine control of free energy changes in electron transfer
processes. It has therefore been ofgreat interest to understand how proteins
influence the chemistry of specific redox sites.
For two chemically identical redox sites, e .g. two hemes, the difference
in redox potential is given by the difference in electrical potential at each
site. This difference could arise from permanent potentials, such as those
from specific liganding groups, charged amino acids, etc, or from potentials
that are induced during the oxidation or reduction process. The latter arise
from the dielectric response of the protein and the surrounding solvent.
Thus differences in redox potentials are determined by the same factors
that determine differences in pK values. The difference in redox potential,
!:J.Em, between two sites is then given by:
22.
!:J.E� is the difference in redox potential of the isolated groups and thus
reflects chemical differences (i.e. a porphyrin vs a chlorophyll). The terms
!:J.!:J.G env and !:J.Gttr have essentially the same meaning as they do in the
discussion of pK values (see Equation 20) and may be calculated in exactly
the same way. As for pK values, !:J.!:J.G env is pH independent, while l1Gttr is
pH dependent because it involves interactions between the redox site and
tit ratable groups. The only difference is that here one compares redox
potentials in two different sites rather than a single ionizable site in macro
molecule and solvent.
Many proposals have emerged regarding mechanisms used by proteins
to control redox potentials, and Equation 22 clearly shows that this control
can be achieved in many ways [see discussion by Rogers (94)] . Rees (92,
93) has suggested that surface charge plays a central role, thus emphasizing
the last term in the equation. Kassner (5 1) argued that a nonpolar heme
environment was a central factor in some cases, while Stellwagen ( 1 1 1)
demonstrated that the extent of heme exposure to the solvent could be
correlated with redox potentials. Both factors contribute to the 1111Genv
term; a hydrophobic site would have few dipolar groups interacting with
the heme, while an exposed site would allow the charge on the heme to
induce a stronger reaction field from the surrounding solvent. Using PDLD
calculations, Churg & Warshel ( 1 7) demonstrated that the difference
between the redox potentials of cytochrome c and a small heme-containing
peptide formed from cytochrome c resulted from a reduced reaction field
from the solvent in the protein, which is not compensated by permanent
dipoles on the protein.
There is good evidence that ionizable groups also effect redox potentials.
Moore and co-workers (70, 7 1 , 95) have for some time emphasized the
role of the heme propionate in determining the redox potential of the heme
in cytochrome C55 Z . Indeed, the 65-mV ( 1 .5-kcal/mole) shift in potential
upon protonation of the propionate essentially equals the 1 . I-unit shift in
the propionate pK upon oxidation of the heme. These complementary
interactions result from the !:J. G Itr term in Equations 20 and 22. Rogers et al
(95) used Warwicker's finite difference program to calculate the interaction
energy and obtained 90 mV, in reasonably good agreement with the experi-
326 SHARP & HONIG
mental value. Bashford & Karplus ( 1 0) accounted for changes in pK

induced by changes in redox state in azurin with FDPB calculations, and
s. Watovitch & R. Josephs (private communication) have used the method
to study ionic strength effects on redox potentials in cytochrome. Recently,
the PDLD method has been incorporated into a dynamics scheme and
used together with free energy perturbation methods to calculate the effect
of Arg-38 on the redox potentials of the heme in cytochrome c (2 1 ) .
One might expect proteins to exploit all available options i n controlling
redox potentials. The four hemes in the bound cytochrome of the reaction
center of R. viridis provide a dramatic example. The redox midpoints in
this protein range over 400 m V and show a nonmonotonic oscillatory
pattern when plotted as a function of distance from the membrane surface
(26). FDPB calculations have succeeded in reproducing the expcrimental
pattern quite accurately, but only when all sources of variation in the hcme
environments were considered (38) . In order of importance, the controlling

factors appear to be (a) surface charge, which is asymmetrically distributed
on the protein surface; (b) the nature of the axial ligands; (c) heme-heme
interactions; (d) the difference in the solvent reaction field.
Forces in Dynamics Simulations

Molecular dynamics simulations require calculation of the electrostatic
contribution to the force on each atom or group. The major problem in
such simulations concerns describing water and ion screening. As discussed
in the theory section, a full all-atom approach would simply use Coulomb's
law with B = I for each pair of charge-dipole interactions. This approach
encounters a number of problems, however, not the least of which is
computational expense (see above) . Thus, a variety of methods have been
introduced to mimic the dielectric response of the environment. Solvent
screening, as well as screening from the electrons and dipoles in the macro
molecule, have been accounted for with a constant dielectric B > I and
various distant dependent dielectric functions (43, 66) . In DNA simu
lations, repulsions between phosphates have been minimized by omitting
(59) or scaling down ( 1 20) their charges. Jayaram et al (49) suggested an
alternative approach based on empirically determined effective dielectric
constants derived from FDPB calculations. As yet, no one way of repre
senting solvent screening is universally accepted. For a more detailed
discussion of this issue, see the review by Harvey (39) .
An additional problem in many simulations is that solvation energies
are not accounted for. For example, there is generally no energy penalty
associated with burying a charge. Similarly, the fact that intramolecular
hydrogen bond or ion-pair formation occurs at the expense of interactions
of comparable magnitude with water is frequently ignored. In the absence
of a large scale solvent simulation, it is important to develop methods to
account for the solvation term, which can at times be the dominant factor
in conformational energies. It has recently been shown that adding a l/r4
term to each nonbonded interaction is an effective and simple way of
incorporating solvation effects into standard potential functions (34).
Another approach is to merge FDPB calculations with a molecular mech
anics scheme (K. Sharp, unpublished results).
Electrostatic Energies and Stability: Folding, Denaturation,
and Binding
The determination of the electrostatic contribution to macromolecular
folding, conformational stability, and binding energies of biomolecules is
of central importance. Since the net energy associated with these processes
is frequently smaller than many of the components such as the electrostatic
contribution, appropriate comparisons with experiment are often difficult

to make.
For proteins, electrostatic interactions primarily result from the effects
of charged amino acids. Their mutual interaction can be probed by study
ing ionic strength and pH effects on protein stability. Salt can influence
stability in a number of ways, including screening electrostatic interactions
and by binding to specific sites (42, 85) . In a study of ribonuclease A,
reversing the charge by succinylating 1 1 amino groups reduced the stability
by 2-4 kcal/mole (45) . This evidence has been used to argue that charge
charge interactions make only small contributions to conformational ener
gies, since a relatively small change in energy results from a drastic modi
fication in charge. In apparent contradiction, studies on T4 lysozyme (76)
and barnase (99) have indicated that a single charge can have stabilizing
effects on the order of 1 -2 kcal/mole. Interactions between the charge and
a helix dipole may be in part responsible for the effect. Alternatively, in
ribonuclease A, individual electrostatic interactions may be on the order
of I kcal/mole and, due to cancellation, the net electrostatic energy is also
of the same magnitude.
Baldwin and co-workers have been studying the interaction of charged
residues with isolated a-helices in work that promises to reveal a great deal
about electrostatic interactions (e.g. 1 06, 1 07) . The problem is in some
ways more complicated than studying folded proteins since it is necessary
to consider the equilibrium between helical and random states. To study
problems of this type, Vasquez et al ( 122a) have modified Zimm-Bragg
theory so as to incorporate sequence-specific electrostatic effects.
The calculation of binding energies is closely related to the problem of
conformational energies, since both require determination of the change
in total electrostatic energy between initial and final states. In binding, the
328 SHARP & HONIG
relevant electrostatic interactions are the mutual attraction or repulsion

of charged groups balanced by the desolvation penalty associated with
removing these groups from water.
To evaluate the energetic changes associated with a particular process,
it is necessary to begin with an expression for the total electrostatic energy
(e.g. Equation 1 6) . The electrostatic energy that contributes to a process
is obtained from the change in ACt betwecn the initial and final states.
These states might correspond to the native and denatured states for
folding problems, two alternate conformations for model building studies,

a single conformation at different ionic strengths for salt effects, native
and variant proteins for mutagenesis studies, and the separated and bound
states of the ligand-receptor, protein-protein, protein-DNA, etc for
binding. In general, these processes entail changes in the solvation of
charged groups balanced by changes in the interactions with the polar
izable electrons, permanent dipoles, and other charges in the macro

molecule. Thus, conceptually, these problems are identical to the cal
culation of pK values and redox potentials discussed above.
If there is a change in the ionization state of any charged group during
a process then an additional work term results from the take up or release
of protons (42, 64). In their thermodynamic analysis of T4 lysozyme
thermal stability, Hawkes et al (40) showed that the effect may be both
electrostatic or entropic in nature. Similarly, the enthalpic and entropic
contributions of counterion release must be considered in problems associ
ated with binding to DNA (54, 55, 62) .
There have been many theoretical treatments of electrostatic con
tributions to conformational and binding energies based on simplified
models for proteins and DNA (60, 6 1 , 65, 1 1 0) . Few studies, however,
consider detailed atomic structures. Two examples that do are Pack &
Klein's work on the ionic strength dependence of the B to Z DNA tran
sition (54, 86) and the study by Novotny et al (80) of antigen-antibody
binding energies. A recent paper illustrates how the FDPB method can be
used to calculate electrostatic contributions to conformational energies
and binding energies, including solvation and desolvation effects (32) . In
an application to the problem of conformational energies it was found that
solvation energies of charged amino acids are an important component in
distinguishing stable from misfolded structures (32, 79) .
As mentioned in the discussion of forces, most caleulations of binding
energies have dealt only with the charge-chargc interaction component
and ignored changes in solvation energies. Recent FDPB calculations on
the electrostatic interactions of IX-helices in a 4-helix bundle protein showed
that the helix dipoles destabilize the packing in both the antiparallel and
the parallel arrangements (33). The desolvation penalty per helix was about
3 kcal/mole, while the attractive interactions between helix dipoles were
only about - 0.5 kcal/mole, illustrating the crucial importance of account
ing for desolvation in binding calculations.
SUMMARY AND PROSPECTS
In the introductory section of this review, we alluded to the difficulty of

applying electrostatic theory to problems in molecular biophysics. As an
extreme example of this point, most experiments on biophysical systems

are carried out in water, usually at some nonzero ionic strength. However
until recently there have been no adequate methods for treating solvent
effects on anything but highly simplified models of biological macro
molecules. Advances in theoretical and computational methods have
changed this situation dramatically. Indeed, as summarized in the appli
cations section of this article, it is now possible to account for a wide range
of phenomena that are primarily electrostatic in origin and that depend
critically on the details of the three-dimensional structure of a macro
molecule and on the properties of the surrounding solvent. Moreover, with
the availability of accurate experimental probes of these phenomena as
well as detailed structural information, critical testing and refinement of
theoretical methods have become possible. We have attempted to sum
marize the basis for this specific progress and to identify general issues
that arise in applications of electrostatic theory to problems associated
with the structure and function of proteins and nucleic acids.
ACKNOWLEDGMENTS
We thank our colleagues Richard Fine, Richard Friedman, Michael

Gilson, Marilyn Gunner, Arald Jean-Charles, Anthony Nicholls, and An
Suei Yang for many stimulating discussions. This work was supported by
grants from the NIH (GM-305 1 8, GM-4 1 3 7 1 ), the NSF (DMB85-03489),
and the ONR (NOOO I 4-86-K-02 1 83).
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ANNUAL

Theory and Applications

Kim A. Sharp and Barry Honig

Department of Biochemistry and Molecular Biophysics,

KEY WORDS: finite difference Poisson-Boltzmann, macromolecular theory,

Acid-Base Equilibria................................................................................................. 322

PERSPECTIVE AND OVERVIEW

A recent Annual Review describes a performance at the court of Louis XV

areas in molecular biophysics. The fundamentals of classical electrostatics ,

owing to the vast amount of structural information about these macro­

is valid on all scales of biological interest. Of course, the theory must be

The Poisson Equation

which relates spatial variation of the potential ¢ r

When the charge distribution can be described in terms of a set of point

Essentially all electrostatic models used in studying macromolecules are

Poisson and Coulomb equations can be written in the forms

respectively. If the dielectric varies through space, then Coulomb's law is

V' c(r)V¢(r) = -4np(r), 5.

definition of these terms depends solely on the spatial resolution at which

redistribution of charges, such as the rearrangement of mobile ions in ionic

E(q) = qr/r3 and E(p) = :!Tp, 6.

where N is the number density of the polarizable bodies. Although this

resentations. The LDC model effectively distributes the dielectric response

LDC model, each atom is represented as a sphere of constant dielectric,

a) Uniform b) Local c) Indue ible

qi-""i qje ""j

<j:i>/V (E-1)E/4lt E i (1+8mx)V i)

EXPLICIT ATOM REPRESENTATIONS The atoms and molecules in a system

presumed to be treated explicitly, they can be assumed to occur in free

experimentally, so the extent to which all-atom models succeed in repro­

STATISTICAL MECHANICS REPRESENTATIONS The Langevin model The

( 1 4), nor does it properly represent the effect of dielectric saturation or

ments of orientational polarization of polar dielectrics are based on the

potential of each ion is assumed to be uniform throughout solution. The

The previous section summarizes the physical interactions that underlie

where <Pfj (x = c, e, s, or m) is the potential at charge i due to charge j. I

I The complication introduced by the nonlinearity of ¢m is ignored here.

Equation 1 6. The excellent review by Harvey (39) should be consulted for

The Protein Dipole Langevin Dipole Method

coworkers (128, 1 30). The PDLD method treats electronic polarizability

Analytical Solutions to the Poisson-Boltzmann Equation

sidered further here.

counterions around DNA are territorially bound, or condensed. Con­

Numerical Solutions to the Poisson-Boltzmann Equation

lems at the level of individual residues and of phenomena that depend on

nique in which Coulomb's law is used to determine the potentials and

method implicitly assumed a uniform dielectric constant in both solute

The Finite Diffe rence Poisson�Boltzmann Method

DEFINING THE MOLECULAR SURFACE AND ATOMIC CHARGES Most studies

THE INTERNAL DIELECTRIC CONSTANT The assignment of a dielectric con­

the high-frequency dielectric constant of organic liquids, for the entire

explicitly in each conformation and only electronic polarizability need be

NUMERICAL CONSIDERATIONS The choice of lattice size is a trade-off

DYNAMICAL EFFECTS The FDPB method calculates the electrical potential

sites on a protein, it may be necessary to consider whether conformational

Electrical Potentials around Macromolecules

have important effects on electrical potentials that will be lost if simplified

on the association rates of charged molecules. This phenomenon has

considerable experimental and theoretical effort has been invested in recent

flGf = L 3 (¢�+¢;+¢';') = L qi¢�!2. 1 7.

The change in solvation free energy .1 .1 G solv in transferring an ion of radius

.1.1G solv q .1 tjJr q 2 ( 1 /82 - 1/8 1 )

standardly used in intermolecular potential functions (44, M. Karplus,

owing to the vast amount of structural information about these macro

experimentally, so the extent to which all-atom models succeed in repro

counterions around DNA are territorially bound, or condensed. Con

THE INTERNAL DIELECTRIC CONSTANT The assignment of a dielectric con

izable electrons, permanent dipoles, and other charges in the macro