y

polymer made of amino acios questions like

d)
>
-

protein
. His-

QSB #7
Function of proteins
- Rosetta Fold

MBC Chapter 3

until sharper switches

-
Course outline: what, when, and why?

Biochemistry 1 Modeling of biological processes 1 Design 1

introduction, motivation, basic concepts reactions, ODEs, TX/TL, regulation autorepression, toggle switch, AR clock

Biochemistry 2 Modeling of biological processes 2 Design 2

structure, function, reactions, systems post-TX, stochasticity, Gillespie, Langevin chemotaxis, retroactivity

Molecular biology 1 Analysis techniques 1 Design 3

DNA, replication, transcription, translation stability, nullclines, sensitivity, perturbations insulation, shared resources, cell-free extracts

Molecular biology 2 Analysis techniques 2

variations, regulation adaptation, disturbance, 2D limit cycles

Recombinant DNA techniques Analysis techniques 3 Final project

restriction, cloning, libraries nD limit cycles, bifurcation, model reduction write-up, poster, presentation

2
Overview for today
MAIN OBJECTIVE
Get familiar with the fundamental role of proteins

1.How do proteins and ligands interact?

2.What are functional domains and why are they conserved?
3.Why are complementary and modularity important?
4.What does the dissociation constant represent?
5.How can basic enzyme kinetics be modeled?
6.What are the main advantages of multienzyme complexes?
7.What are common strategies for regulating enzyme activity?
3
Recap

4
 conversi
rier, as illustrated in Figure 2–22. Enzymes are among the most effective catalysts unless c
Enzymes lower the activation energy activatio
b) from it
reaction
This ene
an unusu
activation enzyme lowers molecule
energy for activation X Y, t
a reaction energy for much lar
total energy

total energy
Y X catalyzed this reac
reaction
Y Y
d more rar
Y X
always p
b b
total ene
reactant reactant favorable
minus en
X X (B) Energ
can be lo
c c by the lin
product product
particula
(A) (B) greatly re
uncatalyzed enzyme-catalyzed
reaction pathway reaction pathway reactions

5
 able reaction of rock falling has been directly coupled to the energetically unfavor- only. In (B), the same reaction is coupled
able reaction of lifting the bucket of water. Note that because part of the energy is to a second reaction; this second reaction

Coupling favorable and unfavorable reactions

used to do work in Figure 2–32B, the rocks hit the ground with less velocity than in
Figure 2–32A, and correspondingly less energy is dissipated as heat.
is analogous to the synthesis of activated
carrier molecules. The energy produced in
(B) is in a more useful form than in (A) and
Similar processes occur in cells, where enzymes play the role of the paddle can be used to drive a variety of otherwise
wheel. By mechanisms that we discuss later in this chapter, enzymes couple an energetically unfavorable reactions (C).

(A) (B) (C)

hydraulic USEFUL
machines WORK

heat heat

kinetic energy of falling rocks is part of the kinetic energy is used to lift the potential kinetic energy stored in
transformed into heat energy only a bucket of water, and a correspondingly the raised bucket of water can be
smaller amount is transformed into heat used to drive hydraulic machines that
carry out a variety of useful tasks

6
synthesis of nucleic acids (polynucleotides) from nucleoside triphosphates, as

Building polymers by repetitive condensation

illustrated on the right side of Figure 2–43.
Note that the repetitive condensation reactions that produce macromolecules
can be oriented in one of two ways, giving rise to either the head polymerization
HOW CELLS OBTAIN ENERGY FROM FOOD

or the tail polymerization of monomers. In so-called head polymerization, the

reactive bond required for the condensation reaction is carried on the end of the HEAD POLYMERIZATION (e.g., PROTEINS, FATTY ACIDS)
MBoC6 m2.66/2.42
base
3
P P P O 6 + 7
sugar
base
1
OH each monomer carries a high-energy
high-energy intermediate P O
sugar bond that will be used for the
base 6 addition of the next monomer
2
2 ATP
P O
P Pi sugar

H O 7
OH
HOW CELLS OBTAIN ENERGY
2
FROM FOOD
polynucleotide 73
base chain containing
3 2 ADP 2 Pi two nucleotides
P O
sugar products of base
HEAD POLYMERIZATION
ATP hydrolysis
1
(e.g., PROTEINS, FATTY ACIDS) growing polymer, and it must
TAIL POLYMERIZATION therefore
(e.g., DNA, be regenerated each time
RNA, POLYSACCHARIDES)
OH P O
sugar
is added. In this case, each monomer brings with it the reactive
nucleoside base used in adding the next monomer in the series. In tail polymeriza
monophosphate 2
6 + 7 1
bond carried by each monomer is instead + used
7 immediately for
P O
sugar
base
(Figure 2–44).
polynucleotide chain
each monomer carries a3 high-energy We shall see in later chapters that both
each of these
monomer carriestypes of po
containing three nucleotides
bond that will be used for the a high-energy bond
6
P O
addition of the next monomer
sugar
used. The synthesis of polynucleotides
7 and some simple polysa
for its own addition
by tail polymerization, for example, whereas the synthesis of pro
MBoC6 m2.68/
OH head polymerization process.
Figure 2–43 Synthesis of a polynucleotide, RNA or DNA, is a multistep process driven by ATP
7 1
hydrolysis. In the first step, a nucleoside monophosphate is activated by the sequential transfer of
the terminal phosphate groups from two ATP molecules. The high-energy intermediate formed—a Summary 7
 Methionine Aspartic acid acid side
Leucine chains. There are three types of these weak bonds: hydrogen bon
Tyrosine
(Met) (Asp) trostatic attractions, and van der Waals attractions, as explained in Chapt
(Leu) (Tyr)

Proteins are polypeptides

p. 44). Individual noncovalent bonds are 30–300 times weaker than th
covalent bonds that create biological molecules. But many weak bonds
(“water-fearing”), others are negatively or positively charged,
parallel cansome hold tworeadily formof a polypeptide chain tightly together. In
regions
110 Chapter 3: Proteins covalent bonds, and so on. Panel 3–1 (pp. 112–113)the shows their atomic structures
combined strength of large numbers of such noncovalent bonds det
and Figure 3–2 lists their abbreviations. the stability of each folded shape (Figure 3–4).
As discussed in Chapter 2, atoms behave almost as if they were hard spheres
with a definite radius (their van der Waals
Figureradius).
3–1 TheThe requirement
components ofthat no two
a protein.
OH AMINO ACID SIDE CHAIN A
atoms overlap plus other constraintsAlimit theconsists
protein possibleofbond angles in a poly-
a polypeptide
O O peptide chain (Figure 3–3), severely backbone
restrictingwith
the attached
possible side three-dimensional
chains. Each
Aspartic acid Asp D negative Alanin
C arrangements (or conformations) of atoms.
MBoC6 m3.01/3.01 Nevertheless, a long
type of protein differs in its sequence flexible
Glutamicand chain
acid Glu E negative Glycin
polypeptide backbone side chains such as a protein can still fold in an enormous
number ofnumber of ways.
amino acids; therefore, it is the
Arginine Arg R positive Valine
CH2 112 PANELCH The
3–1:
2
folding
Theof20 a protein
Amino chain is sequence
Acids alsoFound
determined
of the by many different
inchemically
Proteins different
Lysine side
sets of Lys K positive Leucin
weak noncovalent bonds that form between chains thatonemakes
part ofeachthe protein
chain and another.
distinct.
H H O H H O Histidine His H positive Isoleuc
O atoms in the polypeptide
These involve Thebackbone,
two ends as of well as atoms chain
a polypeptide in the areamino
amino + carboxyl Asparagine Asn N uncharged polar Proline
terminus H N C C N C C N C C N C
acid side chains.terminus
C
There are three typeschemically
of these weak bonds:
different: thehydrogen
end carrying bonds, theelec-
trostatic attractions, and van der Waals attractions, as explained Glutamine
in written
ChapterNH 2 (see Gln Q uncharged polar Pheny
(N-terminus) (C-terminus) free amino group (NH3+, also 2)
H H H O H p.HACID O
44). Individual noncovalent bondsisare the30–300 times weaker
amino terminus, Serine
than
or N-terminus, the typical Ser S uncharged polar Methio
THE AMINO covalent bonds that create biological and molecules. But many
that carrying
OPTICAL
weak
the free bonds
Threonine
carboxyl
ISOMERS
acting in Thr T
group
The α-carbon
uncharged polar atom is asym
Trypto
CH2 CH2 parallel can hold two regions of a polypeptide chain tightly together. this way, Tyr Y allows for polar
two mirror ima
The general peptide
formula of an amino acid is (COO–, also written COOH) theIncarboxyl
isTyrosine uncharged Cystein
peptide bond
the combined strength of large numbers of such noncovalentThe bonds determines isomers, L and D.
CH2 bonds CH terminus or C-terminus. amino acid
α-carbon
the stability of each folded shape (Figure
atom 3–4).
sequence of a protein is always presented POLAR AMINO ACIDS
S H3C CH3 H in the N-to-C direction, reading from left
Figure 3–2 The 20 amino acids commonly found in proteins. Each am
2 PANEL 3–1: The 20 side Amino
chains Acids Found in Proteins
amino AMINO ACID to
carboxyl right.
SIDE CHAIN AMINO ACID
letter abbreviation. There SIDE CHAIN
C COOH group H are equal numbers of polar and nonpolarHside c
CH3 group H2N here as polar are large enough to have some nonpolar properties (for exa
Aspartic acid Asp D negative Alanine see Panel 3–1
structures, Ala (pp.A112–113).
nonpolar
Glutamic
R acid Glu E negative Glycine + Gly G nonpolar
side-chain group NH3 COO– COO–
Methionine Aspartic acid Leucine Tyrosine Arginine Arg R positive Valine Val V nonpolar MBoC6 m3.02/3.02
THE AMINO ACID (Met) (Asp) (Leu)
R is commonlyOPTICAL
(Tyr)
one of 20 ISOMERS
Lysine Lys The
different side
K αpositive
chains.
L Leucine
-carbon atom is asymmetric, which Leu
C α
L nonpolar Cα
Histidine His allows
H positive Isoleucine
for two mirror images (or stereo-) Ile I nonpolar
The general formula of an amino acid is At pH 7 both the amino and carboxyl
Asparagine groups
Asn N uncharged polar Proline Pro P nonpolar
are ionized. isomers, L and D.
(“water-fearing”), others are negatively or positively charged, someGlutamine
α-carbon atom readily formGln Q uncharged polar Phenylalanine Phe F nonpolar
H H
Serine Ser S uncharged polar Methionine R
Met M nonpolar R
covalent bonds, and so on. Panel 3–1 (pp. 112–113) shows their +
atomic structures
amino carboxyl Threonine Thr T uncharged polar Tryptophan Trp W nonpolar
and Figure 3–2Hlists
N their
C abbreviations.
COOH H 3 N C COO H H
group 2 group Tyrosine Tyr Y uncharged polar Cysteine Cys C nonpolar
As discussed in Chapter 2, atoms behave almost as if they were hard spheres
with a definite radius R(their van der Waals radius). The requirement R that noPOLAR two AMINO– ACIDS Proteins consist exclusively
NONPOLAR AMINO ACIDS of L-amino acids.
side-chain group NH3+ COO COO– NH3+
atoms overlap plus other constraints limit the possible bond angles in a poly-
Figure 3–2 The 20 amino acids commonly found in proteins. Each amino acid has a three-letter and a one- 8
peptide chain (Figure 3–3), severely restricting the possibleLthree-dimensional C There are equal numbers of polar and nonpolar
letter abbreviation. C D
side chains; however, some side chains listed
Twisting
114 and turning
Chapter 3: Proteins means proteins are folded

unfolded polypeptide Figure 3–5 How a pro

compact conformatio
acid side chains tend t
of the protein, where th
water; the nonpolar am
are buried on the insid
packed hydrophobic c
nonpolar polar polypeptide are hidden from water.
side chains side backbone drawing, the protein co
chains 35 amino acids.

polar side chain on the hydrophobic core region

outside of the molecule contains nonpolar
can form hydrogen side chains
bonds to water

folded conformation in aqueous environment

9
 Structure
120
matters
Chapter 3: Proteins
more than sequence
120 Chapter 3: Proteins

(A) (B)
(A) helix 2 (B)
helix 2

helix 3
helix 3 helix 1
helix 1
NH2
COOH
NH2
COOH

(C)

(C)
yeast
G H R F T K E N V R I L E S W F A K N I E N P Y L D T K G L E N L MK N T S L S R I Q I K NWV S N R R R K E K T I
H2N yeast COOH
R T A F S S E O L A R L K R E F N E N - - - R Y L T E R R R QQ L S S E L G L N E AQ I K I WF QN K R A K I K K S
G H R F T K E N V R I L E S W F A K N I E N P Y L D T K G L E N L MK N T S L S R I Q I K NWV S N R R R K E K
HDrosophila
2N
R T A F S S E O L A R L K R E F N E N - - - R Y L T E R R R QQ L S S E L G L N E AQ I K I WF QN K R A K I K
Figure 3–13 A comparison of a class of DNA-binding domains, called homeodomains, in a pair of proteins from 10
Drosophila
 each organism whose
me contains the DNA
More
otein kinasecomplex
domains, organisms have more complex proteins
120 SH2 domains. In
consist of two or more
edly in the same rela- yeast
families are common
e two-domain combi- Ep1 PHD PHD Ep2
st proteins containing
domain shuffling rel- worm

Ep1 PHD PHD Ep2 Br

Proteins,
human

Znf Ep1 PHD PHD Ep2 Br BMB

surprising, because it
protein-coding genes.
ore complex than the Figure 3–17 Domain structure of a group
11
 The observation that helices occur commonly in biological structures holds
true whether the subunits are small molecules linked together by covalent bonds
Proteins also form macrostructures
(for example, the amino acids in an α helix) or large protein molecules that are
linked by noncovalent forces (for example, the actin molecules in actin filaments).
g This
site is
can (A) A helix is an unexceptional structure, and it is generated
not surprising. actin molecule
h simply
two by placing manyfree similar subunitsassembled
next to each other, each in the same MBoC6 m3.24/3.20
minus end
o strictly
bindingrepeated relationship
subunits to the one structures
before—that is, with a fixed rotation fol-
lowed by a fixed translation along the helix axis, as in a spiral staircase.
n subunits dimer
Many Protein Molecules Have Elongated, Fibrous Shapes
binding
C, see
site proteins: even though many are large and compli-
Enzymes tend to be globular
cated, with multiple subunits, most have an overall rounded shape. In Figure 3–21,
(B)
we saw that a globular protein can also associate to form long filaments. But there
helix
are also functions that require each individual protein molecule to span a large
distance. These proteins generally have a relatively simple, elongated three-di-
mensional structure and are commonly referred to as fibrous proteins.
One large family of intracellular fibrous proteins consists of α-keratin, intro-
e (Figure
duced when we presented binding
the α helix, and its relatives. Keratin filaments are
ced from stable and are the main component in long-lived structures such as
extremely sites
tein horn, and nails. An α-keratin molecule is a dimer of two identical subunits,
hair, that
37 nm
(C) of each subunit forming a coiled-coil (see Figure 3–9). The
with the long α helices
ment sys- regions are capped at each end by globular domains containing bind-
coiled-coil
ing sites. This enables this class of protein to assemble intoring
ropelike intermediate
xfilaments—an
such a important component of the cytoskeleton that creates the cell’s
internal structural framework
binding (see Figure 16–67).
plus end

are often
Fibrous proteins sites
are especially abundant outside the cell, where they are a (A) 50 nm (B)
main component of the gel-like extracellular matrix that helps to bind collections
subunits Figure 3–21 Actin filaments. 12
 that restrict diffusion. For example, the abundant nucleoporins that coat the inner Figure
Proteins do have structure and unstructure both
surface of the nuclear pore complex form a random coil meshwork (Figure 3–24)
that is critical for selective nuclear transport (see Figure 12–8).
for intr
sequen
of polyp
sites fo
binding
they ar
free-en
P unfolde
reversib
+
be eas
P their bi
P therefo
proces
of prote
P (C) Uns
“tethers
P
domain
P networ
a diffus
(A) BINDING (B) SIGNALING (C) TETHERING (D) DIFFUSION BARRIER for the

13
Sometimes non-covalent bonds are insufficient

14
 (TBSV) shown here, for example, is a
mple viruses, which takesshell
thedomain
form of a hollow sphere based on an icosahedron spherical virus about 33 nm in diameter
Figure 3–27). Capsids are often made of hundreds of identical protein subunits formed from 180 identical copies of a

Examples of macrostructures: capsid of viruses

hat enclose and protect the viral
connecting nucleic acid (Figure 3–28). The protein in such
arm
386-amino-acid capsid protein plus an
capsid must have a particularly adaptable structure: not only must it make RNA genome of 4500 nucleotides. To
construct such a large capsid, the protein
everal different kinds of contactsdomain
RNA-binding to create the sphere, it must also change this must be able to fit into three somewhat
rrangement to let the nucleic acid out to initiate viral replication once the virus THE SHAPE AND STRUCTURE OF PROTEINS
different environments. This requires three
as entered a cell. free slightly different conformations, each of
dimers which is differently colored in the virus
particle shown here. The postulated Figure 3–28 The stru
Many 128
Structures in Cells
Chapter Are Capable of Self-Assembly
3: Proteins pathway of assembly is shown; the precise
three dimers
virus. In viruses, man
three-dimensional structure has been free dimers protein subunit often
The information for forming many of the complex assemblies of macromolecules to create a spherical s
determined by x-ray diffraction. (Courtesy
n cells must be contained in the subunits themselves, because purified subunits ofSingle
Steve Harrison.) This capsid encloses
Figure 3–26 protein subunits form dimer
an spontaneously assemble into the final structure under the appropriate con- composed of either R
hexagonally protein assemblies that feature multiple
itions. The first large macromolecular aggregate shown to be capable of self-as- incomplete Figure 3–27). For geo
packed protein–protein contacts. Hexagonally particle
embly from its component parts was tobacco mosaic virus (TMV ).sheet This virus is more than 60 identica
packed globular protein subunits are
viral RNA together in a precisely
long rod in which a cylinder of protein is arranged around a helical RNA core shown here forming either flat sheets or
slight irregularities are
Figure 3–29). If the dissociated RNA and protein subunits are mixed together in tubes. Generally, such large structures are
more subunits can be
subunit not considered to be single “molecules.”
olution, they recombine to form fully active viral particles. The assembly process Instead, like the actin filament described
a larger capsid that re
unexpectedly complex and includes the formation of double rings of protein, symmetry. The tomat
previously, they are viewed as assemblies projecting domain
(TBSV) shown here, fo
hich serve as intermediates that add to the growing viral coat. formed of many different molecules.
tube shell domain spherical virus about
Another complex macromolecular aggregate that can reassemble from its formed from 180 iden
omponent parts is the bacterialcapsid ribosome.
protein This structure is composed of about intact virus
connecting arm
386-amino-acid caps
5 different protein molecules andmonomer 3 different rRNA molecules. Incubating a mix- particle RNA genome of 4500
shown as (90 dimers) construct such a larg
ure of the individual components ribbon under appropriate conditions in a test tube RNA-binding domain
must be able to fit into
auses them to spontaneously re-form modelthe original structure. Most importantly, different environments
uch reconstituted ribosomes are able to catalyze protein synthesis. As might be 10 nm free slightly different confo
20 nm
xpected, Some protein subunits
the reassembly of ribosomesassemble intoa flat
follows sheets
specific in which
pathway: thecertain
after subunits are dimers which is differently co
roteins arranged in hexagonal
have bound to the RNA,patterns. Specialized
this complex membrane
is then recognized proteins
by otherare pro-
sometimes Figure 3–27 The protein capsid of a particle shown here. T
eins, andarranged thissimplest
so on,the
until way in case,
lipid abilayers.
the structure is With
complete.
long core a slight
protein change
or other in the geometry
macromolecule of the
provides virus. The structure of the simian virus
a scaffold pathway of assembly
SV40 three-dimensional stru
It isindividual subunits,
still notthat
clear a hexagonal
how some
determines the sheet
of extent
the more can be converted
elaborate
of the final into
Thisaistube
self-assembly
assembly. (Figure 3–26)
processes
the mechanism thatcapsid
deter-has been determined by x-ray determined by x-ray d
crystallography and, as
MBoC6 for the capsids of
m3.30/3.26
or, withMany
re regulated. more changes,
minesstructures
the lengthinto
in ofa hollow
the cell,
the TMV sphere.
for Protein
example,
particle, seem
where tubes
to and
RNAaspheres
thehave precisely
chain that bind
provides manytheother
core.
viruses, it is known in atomic of Steve Harrison.)
efinedspecific RNA
length that isand
many
Similarly, DNA coremolecules
a times protein in their
than thatinterior
greaterinteracting of form the
theirm3.31/3.27
with
MBoC6
component
actin coats
is thought oftoviruses.
macromol-
determinedetail.
the length
(Courtesy of Robert Grant, Stephan
cules. How The formation
such
of length
the thin offilaments
closed structures,
determination such is
is achieved
in muscle. asinrings,
manytubes,
casesor spheres, In
a mystery. provides
Crainic, and James M. Hogle.)
additional stability because it increases the number of bonds between the protein
MBoC6 m3.29/3.25
subunits. Moreover, because such a structure is created by mutually dependent,
cooperative interactions between subunits, a relatively small change that affects
each subunit individually can cause the structure to assemble or disassemble.
These principles are dramatically illustrated in the protein coat or capsid of many
simple viruses, which takes the form of a hollow sphere based on an icosahedron
(Figure 3–27). Capsids are often made of hundreds of identical protein subunits
that enclose and protect the viral nucleic acid (Figure 3–28). The protein in such capsid protein intact virus
monomer particle
a capsid must have a particularly adaptable structure: not only must it make shown as (90 dimers)
(A) (B)
several different kinds of contacts to create the sphere, it must also change this50 nm ribbon
arrangement to let the nucleic acid out to initiate viral replication once the virus model

has entered a cell.
Many Structures in
Figure 3–29 The structure of tobacco mosaic virus (TMV). (A) An electron micrograph of the viral particle, which consists of 10 nm
a single long RNA molecule enclosed in a cylindrical protein coat composed of identical protein subunits. (B) A model showing
part of the structure of TMV. A single-stranded RNA molecule of 6395 nucleotides is packaged in a helical coat constructed 15
Cells Arecopies
from 2130 Capable ofprotein
of a coat Self-Assembly
158 amino acids long. Fully infective viral particles can self-assemble in a test tube from
 Function
of proteins

16
to destroy a binding site on the surface.
Figure 3–38 The binding site of a
protein. (A) The folding of the polypeptide

Specific binding to ligands via non-covalent bonds

The Surface Conformation of a Protein Determines Its Chemistry
The impressive chemical capabilities of proteins often require that the chemical
chain typically creates a crevice or cavity on
the protein surface. This crevice contains a
set of amino acid side chains disposed in
groups on their surface interact in ways that enhance the chemical reactivity of such a way that they canPROTEIN FUNCTION
form noncovalent
one or more amino acid side chains. These interactions fall into two main cate- bonds only with certain ligands. (B) A
close-up of an actual binding site, showing
gories.
the hydrogen bonds and electrostatic
First, the interaction of neighboring parts of the polypeptide chain may restrict interactions formed between a protein and noncovalent bonds
the access of water molecules to that protein’s ligand-binding sites. Because water its ligand. In this example, a molecule of
molecules readily form hydrogen bonds that can compete with ligands for sites cyclic AMP is the boundligand
ligand.

amino acid
side chains binding
site

H serine (B)
C
CH2
O N hydrogen bond
H O (A) protein
C
H H
C 5′
O O cyclic AMP The region of a protein that associates with a ligand, know
unfolded protein (CH2)3
NH P O ing site, usually consists of a cavity in the protein surface fo
+
FOLDING C NH2 O O ′
3
serine arrangement
H of amino acids. These amino acids can belong
arginine N N H ofCthe polypeptide chain that MBoC6 m3.36/3.33
are brought together when the
NH2 CH2
binding site O O
H H 3–38). Separate regions of the protein surface generally pro
N H N N different ligands, allowing the protein’s activity to be regul
_ N
H later. And other parts of the protein act as a handle to posit
O
O H O cell—an example is the SH2 domain discussed previously,
threonine
electrostatic C
attraction CH protein containing it to particular intracellular sites in respo
CH2 H3C C Although the atoms buried in the interior of the protein h
CH2 glutamic with
H the ligand, they form the framework that gives the sur
folded protein acid its chemical and mechanical properties. Even small changes
(A) (B)
C
the interior of a protein molecule can change its three-dimen
H to destroy a binding site on the surface.
17
The Surface Conformation of a Protein Determine
 Functional domains are evolutionary conserved
FUNCTION PROTEIN FUNCTION 137

polypeptide ligand polypeptide

phosphotyrosine pho

(A) FRONT (A) FRONT BACK BACK(B) FRONT (B) FRONT

the recognition
SH2 family of peptide large SH2 family of peptide
domains. recognition
Mutation domains.
is a random Figure 3–40
pro- Mutation is a The evolutionary
random Figure 3–40 The evolutionary
pro- trace
cess; survival
ival is not. Thus, natural is (random
selection not. Thus,mutation
natural selection
followed(random method followed
by non- mutation applied to by
thenon- method applied to the SH2 d
SH2 domain.
(A) Front and back views of a s
(A) Front and back views of a space-
random
urvival) produces the survival)
sequence producesby
conservation the sequence conservation
preferentially eliminat- byfilling
preferentially eliminat- 18
filling model of the SH2 domain
model of the SH2 domain, with
 matching of one rigid surface with that of another (Figure 3–41C). Such interac-
Complementary binding of proteins
tions can be very tight, since a large number of weak bonds can form between two
surfaces that match well. For the same reason, such surface–surface interactions
can be extremely specific, enabling a protein to select just one partner from the
many thousands of different proteins found in a cell.

string Figure 3–41 Thr

surface proteins can bin
surface 1 the interacting pa
are shown. (A) A
surface 2 protein can bind
polypeptide chain
second protein. (
helix 2 helix 1
bind together to
(C) Two complem
often link two pro
interactions can a
(A) SURFACE–STRING (B) HELIX–HELIX (C) SURFACE–SURFACE
β strands (see, fo

MBoC6 m3.40/3.37
19
Antibodies
138
use versatile and specific loops
Chapter 3: Proteins

heavy chain

VH VH hypervariable loops

NH2

S
S

S
CH1 CH1
S

S
S

S
S

S
VL VL

S
S S
S

S
S

S
S S S

S
S
CL CL

S S
CH2 S S variable domain
of light chain (VL)
disulfide
C H2 bond

CH3 S S
S S CH3 Figure 3–42 An antibody molecule.
A typical antibody molecule is Y-shaped
(A) and has two identical binding sites for
its antigen, one on each arm of the Y. As
constant domain explained in Chapter 24, the protein is
of light chain (CL) composed of four polypeptide chains (two
COOH identical heavy chains and two identical
and smaller light chains) held together
(B) by disulfide bonds. Each chain is made
up of several different immunoglobulin 20
 rium, in which the number of binding (association) even
equal to the number of “unbinding” (dissociation) event

Dissociation constant captures strength of binding

From the concentrations of the ligand, antibody, and a
at equilibrium, we can calculate a convenient measure
ing—the equilibrium constant (K)—(Figure 3–44A). Thi
in detail in Chapter 2, where its connection to free energ
(see p. 62). The equilibrium constant for a reaction in wh
PROTEIN FUNCTION 139 (AB) has unit
B) bind to each other to form a complex
of the binding sites will be occupied by ligand when th
(in moles/liter) reaches a value that is equal to 1/K. Thi
larger the greater the binding strength, and it is a direct
ergy difference between the bound and free states (Figu

B B 1
dissociation
A B A + B
A the surfaces of molecules A and B,
dissociation rate = dissociation × concentration
and A and C, are
rate a poor match
constant of AB and
A
are capable
dissociation rate = kof forming
only a few
off [AB]
weak bonds; thermal motion rapidly
2
breaks them association
apart
A + B A B

association rate = association × concentration × concentration

rate constant of A of B
A A C A
association rate = kon [A] [B]
C
3
AT EQUILIBRIUM:
molecule A randomly encounters
association rate = dissociation rate
other molecules (B, C, and D) thekonsurfaces
[A] [B] =ofkoffmolecules
[AB] A and D
D match
[AB] well
kon and therefore can form
= = K = equilibrium constant
A enough
[A][B] kweak
off
bonds to withstand
A thermal jolting; they therefore
D (A) stay bound to each other

We can measure the strength with which any two molecules bind to each Figure 3–43 How noncovalent bonds
MBoC6
other. As an example, consider a population of identical antibody molecules that mediate interactions between
suddenly encounters a population of ligands diffusing in the fluid surrounding macromolecules (see Movie 2.1). 21
 any enzymes have only one substrate, which they bind and At this steady state, [ES] is nearly co
en process to produce products according to the scheme
k1
Basic enzyme
k
kinetics
utlined in Figure 3–50A. In this case, the reaction is written as
1
rate of ES breakdown
+ kcat
[E][S] =
k–1 +kkcat
[Eo] – [ES] [S]
k–1 [ES] + kcat [ES]
=
1 kcat
E+S ES E+P
and defining the constant k
Km–1as
or, since the concentration of the f
k–1 + kcat
ere we have assumed that the reverse reaction, in which E + P to [Eo] – [ES],
combine to k1 form EP and then ES, occurs so rarely that we can
nore it. In this case, EP need not be represented, and we can
xpress the rate of the reaction—known as its velocity, k1
PROTEIN FUNCTIONV, as [ES] = [E][S] =
[Eo][S]
S] = k–1 + kcat
Km + [S] V = kcat [ES] Vmax

here [ES]
V =iskcat
the[ES],
concentration of the enzyme–substrate complex, Rearranging, and defining the con

rate of reaction
ing that we obtain the famous
nd kcat
nten is the turnover number, a rate constant that has a value
equation
qual to the number of substrate molecules processed per 0.5Vmax k–1 + kcat
nzyme molecule kcat [Eeach second. k1
o][S]
But how V =does the value of [ES] relate to the concentrations that
Km + [S]
e know directly, which are the total concentration of the we get
Km substrate concentration 22
nzyme, [E ], and the concentration of the substrate, [S]? When
 this enzymatically catalyzed reaction, which occurs millions of times faster than
uncatalyzed hydrolysis.

Example: hydrolysis of polysaccharides by lysozyme Other enzymes use similar mechanisms to lower activation energies and
speed up the reactions they catalyze. In reactions involving two or more reactants,
the active site also acts like a template, or mold, that brings the substrates together
in the proper orientation for a reaction to occur between them (Figure 3–52A). As
we saw for lysozyme, the active site of an enzyme contains precisely positioned

+
+
asing activation energy
for uncatalyzed reaction
146 Chapter 3: Proteins
zed
ST ES EP E+P (B)
(A)
SUBSTRATE S + E PRODUCTS
This substrate is an oligosaccharide of six sugars, The final products are an oligosaccharide of four sugars
labeled A through F. Only sugars D and E are shown in detail. (left) and a disaccharide (right), produced by hydrolysis.

yme– R OH The reaction catalyzed by lysozyme. (A)RThe enzyme lysozyme

CH23–50
Figure H H
CH2OH (E) catalyzes t
A AB C chain, which
O is its
O
Fsubstrate (S). The enzyme
A B C first binds to the chain to form Oan enzyme–subs
O
F
O E O E
D O D O O
EST O catalyzes the cleavage of a specific covalent bond in the O backbone of the polysaccharide, fo

(EP) that rapidly dissociates. Release of the severed chain (the products P) leaves the enzym
energy

CH2OH R side chain CH2OH R

S on sugar E
molecule. (B) A space-filling model of the lysozyme molecule bound to a short length of polys
, the (Movie 3.8). (B, courtesy of Richard J. Feldmann; PDB code: 3AB6.)
B C C C
ergy ES P ES Glu35
C O
Glu35
C O
Glu35
C O
EP
O H O H O O
EP H H
progress HOCH2 CH2OH HOCH2 CH2OH MBoC6HOCH
m3.50/3.46 CH2OH
H 2 H H
of reaction O D O EO O O D O O EO O O D O O O E
O O
O
activation energy R
C
R
C
R
C
ding for catalyzed reaction H C1 carbon
R
O
R
H
R

09 to O O C O
O C C O C
isely C
Asp52 Asp52
C
Asp52

pate 23
 interm
It is a
Tightly enzyme
Common Bound Smallstrategies
Molecules Add Extra Functions to Proteins interm
Although we have emphasized the versatility of enzymes—and proteins in gen- coval
panel
eral—as chains of amino acids that perform remarkable functions, there are many subst
instances in which the amino acids by themselves are not enough. Just as humans al., N

+
–
–
+

Figur
enzym
(A) enzyme binds to two (B) binding of substrate (C) enzyme strains the toget
substrate molecules and to enzyme rearranges bound substrate
orients them precisely to electrons in the substrate, molecule, forcing it
stabil
encourage a reaction to creating partial negative toward a transition (C) Ap
occur between them and positive charges state to favor a reaction subst
that favor a reaction reacti

24
Multienzyme complexes increase efficiency
PROTEIN FUNCTION 149

FATTY ACID SYNTHASE acyl carrier

domain

N C

2 1 4 5 3 termination
domain (TE)
enzyme domains
(A)

1
TE
(D) 20 nm

PYRUVATE DEHYDROGENASE COMPLEX

1

4 2
5 2

1
2

4
3
(B) 5 nm (C) etc. (E)
25
Figure 3–54 How unstructured regions of polypeptide chain serving as tethers allow reaction intermediates to be
 usually multiple points of control by different final products, each of which works
to regulate its own synthesis (Figure 3–56). Feedback inhibition can work almost

Feedback regulation of enzymes

instantaneously, and it is rapidly reversed when the level of the product falls.

aspartate

aspartyl
phosphate

egulate when A B C aspartate

semialdehyde

ls how many
the gene that
homoserine
ols enzymatic X
lysine
ompartments, negative
artment (dis- regulation
Figu
n scaffold (see Y
threonine In th
bios
are also cova- ami
indic
on by targeted bac
con
m (see Figure own
Z
rates through leve
dan
o the specific prod
initia
Figure 3–55 Feedback inhibition of a synt
single biosynthetic pathway. The end enz
ds a molecule product Z inhibits the first enzyme that is
methionine isoleucine
inhib

e site, thereby unique to its synthesis and thereby controls

cts. For exam- its own level in the cell. This is an example 26
 Symmetric Protein Assemblies Produce Cooperative Allosteric
Transitions
Allosteric regulation via conformation changes A single-subunit enzyme that is regulated by negative feedback can at most
decrease from 90% to about 10% activity in response to a 100-fold increase in
the concentration of an inhibitory ligand that it binds (Figure 3–59, red line).
Responses of this type are apparently not sharp enough for optimal cell regulation,
and most enzymes that are turned on or off by ligand binding consist of symmet-
ric assemblies of identical subunits. With this arrangement, the binding of a mol-
ecule of ligand to a single site on one subunit can promote an allosteric change in
the entire assembly that helps the neighboring subunits bind the same ligand. As
152 Chapter 3: Proteins a result, a cooperative allosteric transition occurs (Figure 3–59, blue line), allowing

INACTIVE Figure 3–57 Positive

ACTIVE regulation caused
by conformational coupling between
two separate binding sites. In this
molecule example, both glucose and molecule X molecule
X X
bind best to the closed conformation of a
protein with two domains. Because both
molecule
glucose and Xmolecule X drive the protein
toward its closed conformation, each
ligand helps the other to bind. Glucose
molecule positive negative
glucose and molecule X are therefore
glucose said to bind regulation
X regulation
cooperatively to the protein.

ACTIVE INACTIVE
10% active 100% active 100% active 10% active

The relationships shown in Figures 3–57 and 3–58 apply to all proteins, and
they underlie all of cell biology. The principle seems so obvious in retrospect
that we now take it for granted. But the discovery of linkage in studies of a few
MBoC6 m3.58/3.53
enzymes in the 1950s, followed by an extensive analysis of allosteric mechanisms MBoC6 m3.59/3.54
in proteins in the early 1960s, had a revolutionary effect on our understanding of
27
biology. Since molecule X in these examples binds at a site on the enzyme that
 3–60. In this view, ligand binding perturbs an all-or-none equilibrium between
these two states, thereby changing the proportion of active molecules. Both mod-
Cooperativity makes sharper switches els represent true and useful concepts.

Many Changes in Proteins Are Driven by Protein Phosphorylation ENZYME ON

Proteins are regulated by more than the reversible binding of other molecules. A
second method that eukaryotic cells use extensively to regulate a protein’s func-
EIN FUNCTION tion is the covalent addition of a smaller molecule to one or more of its amino acid 153
side chains. The most common such regulatory modification in higher eukaryotes inhibitor
is the addition of a phosphate group. We shall therefore use protein phosphoryla- DIFFICULT
100 tion to illustrate some of the general principles involved in the control of protein
Figure 3–59 Enzyme activity versus TRANSITION
function through the modification of amino acid side chains. the concentration of inhibitory ligand substrate
A phosphorylation event can affect the protein that is modified in three
for single-subunit and multisubunit
important ways. First, because each phosphate group carries two negative
allosteric enzymes. For an enzyme with a
percent enzyme activity

charges, the enzyme-catalyzed addition of a phosphate group to asingle protein can(red line), a drop from 90%
subunit
cause a major conformational change in the protein by, for example,
1 subunit enzyme activity
attracting a to 10% activity (indicated
50 cluster of positively by the
charged amino acid side chains. This can, in turn,
2 subunits two the
affect dots on the curve) requires a
binding of ligands elsewhere on the protein surface, dramatically 100-fold
4 subunits changingincrease
the in the concentration of
inhibitor. The enzyme activity is calculated
Figure 3–60 A cooperative allosteric transition in an enzymefrom the simple
composed of equilibrium relationship EASY
two identical subunits. This diagram illustrates how the conformationK = [IP]/[I][P], where P is active protein, TRANSITION
of one
subunit can influence that of its neighbor. The binding of a singleI is molecule
inhibitor,of and IP is the inactive protein
0 an inhibitory ligand
5 (yellow) to one subunit of the enzyme 10occursbound
with difficulty
to inhibitor. An identical curve
because it changes the
inhibitor concentration conformation of this subunit and thereby disrupts the
applies to any simple binding interaction
symmetry of the enzyme. Once this conformational change has occurred,
between two molecules, A and B. In
however, the energy gained by restoring the symmetric pairing interaction
vely small change in ligand concentration
between the two insubunits
the cell to itswitch
makes especiallytheeasywhole contrast,
for the second subunit a multisubunit allosteric enzyme
canchange.
respond in a switchlike manner to a
bly from an almost fully active toto
anbind the inhibitory ligand and undergo the same conformational
almost fully inactive conformation (or
Because the binding of the first molecule of ligand increases thechange in ligand concentration: the steep
affinity with
rsa). which the other subunit binds the same ligand, the response of response
the enzymeistocaused by a cooperative
changes in the concentration of the ligand is much steeper thanbinding
the response ENZYME OFF
principles involved in a cooperative
MBoC6
“all-or-none”
m3.60/3.55
transition are the same of the ligand molecules, as
of an enzyme with only one subunit (see Figure 3–59 and Movie 3.10). 28
proteins, whether or not they are enzymes. Thus, for example, they are crit- explained in Figure 3–60. Here, the green
Overview for today
MAIN OBJECTIVE
Get familiar with the fundamental role of proteins

1.How do proteins and ligands interact?

2.What are functional domains and why are they conserved?
3.Why are complementary and modularity important?
4.What does the dissociation constant represent?
5.How can basic enzyme kinetics be modeled?
6.What are the main advantages of multienzyme complexes?
7.What are common strategies for regulating enzyme activity?
29