A Solvation Potential with Improved Contact
Definitions and Optimized by Extensive Threading
Alan A. Dombkowski and Gordon M. Crippen2
Biophysics ResearchDivision, University of Michigan, Ann Arbor, MI 48109-1055,USA.
*College of Pharmacy,University of Michigan, Ann Arbor, MI 48 109-1065,USA.
Protein structural knowledge is essentialto understanding
the molecular basis of disease. Drug design and discovery
are facilitated by understandingthe three dimensional
shapeof relevant proteins, as will be future modesof
diseaseintervention such as genetherapy. While
identification of diseaserelated protein sequenceshas
dramatically increasedthrough genomemapping efforts,
the rate of determination of the associatedstructures
remains relatively slow. Despite recent advancements,
experimental methodsof protein structure determination
remain time consuming and labor intensive. Therefore,
computational methodswhich can predict the correct
protein structure for a given amino acid sequencewould be
a tremendousaid in biomedical research. Homology
modeling is precluded if the sequenceof interest haslow
similarity to proteins of known structure. For these
sequences,structure prediction requires an energy potential
which can identify the native structure out of any number
of alternative conformations. We have developeda
solvation energy potential which is optimized to identify
the native structure as the lowest in energy. The potential
has few adjustableparameterswhich are trained so the
Protein Data Bank (PDB) structure for eachprotein in a
training set is the lowest in energy when comparedto a
very large set of alternatives - over lo5 alternativeson
average. Intramolecular contact definitions are obtained
for all possible amino acid pairs from a survey of protein
structuresand are continuous as a function of interresidue
distance. Training with only 25 native proteins produces
an energy potential which is quite successfulin structure
identification tests. Small proteins and those with
prosthetic groups posethe greatestchallenge.
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145
Introduction
Developmentof a potential function requires consideration
of the method usedto produce the alternative structures.
All-atom representationsof proteins provide the greatest
detail; however, they are computationally demandingto
generate,resulting in reducedcoverageof conformation
space. A method which provides a wider sampleof
possible conformations with less atomic detail is threading
(Hendlich et al., 1990;Crippen, 1991). Threading uses
backbonesegmentsfrom known PDB structuresto
produce alternativeswhich have protein-like
characteristicssuch asproper bond lengths, angles and
commonly observedsecondarystructure. For a query
sequenceof length N, any contiguous structural segmentof
N residuestaken from a PDB structure of length N or
greatercan be evaluatedas a possible structure for the
query sequence.However, since the amino acid side
chains of the query sequencediffer from those of the
structural templatesbeing evaluated,compatibility is
obtainedby representingeach side chain as a point in
spacesuch as the p carbon or a centroid (Bryant &
Lawrence, 1993), which then takeson the identity of the
respectiveside chain in the query sequence.Threading
provides a computationally efficient method to samplea
large region of conformation space,but the trade-off is a
loss of atomic detail in the side chains.
Most potentials are a function of inter-side chain and side
chain-backboneinteractions. The potential is constructed
such that eachdefined interaction has an associatedenergy
parameterwhich may be obtained by statistical analysis of
observedinteractions in known PDB structures(Sippl,
1990;Miyazawa & Jemigan, 1985) or optimization
methods(Maiorov & Crippen, 1992; Goldstein et a1.,1992;
Huber & Torda, 1997; Chiu & Goldstein, 1998). Many
functional forms have beentried, most with large setsof
energy parameters.However, in an assessmentof
statistical potentials Thomas and Dill concluded, most of
the information about protein energeticscontained in
complex statistical potentials is simply hydrophobic
clustering propensity . . .(Thomas & Dill, 1996).
Hydrophobic burial is inversely related to solvent
exposure,and as noted by Jones,By far the largest
componentof any useful fold-recognition potential are
going to be the solvation components (Jones,1997).
It is evident that solvation potentials which can evaluate
simplified protein structures,such as those generatedvia
threading methods,would be of great advantage. The
challenge in constructing this type of potential lies in the
loss of side chain atomic detail. The extent of solvent
exposure,or conversely burial, of a residue in a given
protein structure is a function of atomic contact with other
side chains. In current threading methods,gappedor
ungapped,the simplified representationof eachside chain
as a point in spaceintroduces error when assessing
inter-residuecontactsand the extent of solvation.
Huang et al. (1995) demonstratedthat a simple potential
consisting of only a hydrophobic fitness score(HF)
reflecting the extent of burial of apolar amino acids could
successfully identify the correct structure for most
sequencesin their test set. Their HF potential achieveda
remarkable 85% accuracy of native structure identification
in an ungappedthreading test. The results clearly
demonstratethe strength of constructing a potential based
on the hydrophobic effect; however, a problem in
simplified potentials is also revealed. In the HF potential a
contact is defined as any two side chain representations
which are within 7.3 A of eachother. The simple on/off
form of a contact definition is computationally appealing
and is commonly used; however, discontinuous contacts
(hard cutoffs) may diminish the accuracyof a potential and
preclude the use of energy minimizers which require
smooth and differentiable functions. In a test of six
potential functions where decoy structureswere produced
by a four stateoff-lattice model, Park and Levitt (1996)
concluded that functions which are distancedependent(in
more than an on/off sense)are more effective than those
which are not. In other studies it was found that the hard
cutoff usedin the I-IF potential contributed to incorrect
structure identifications when decoys were generatedby
molecular dynamics simulations (Huang et al., 1996;Park
et al., 1997). One problem noted with sharpcutoffs is that
slight changesin atomic position may result in large
changesin contacts. Here, we seekto continue the focus
on solvation while improving on the treatmentof contacts
in simplified models of proteins.
Contact Definitions
A contact is typically defined as two side chains (Cp or
centroid) being within somespecified cutoff distancefrom
146
eachother. There is much variation in the cutoff distances
usedin energy potentials, with a typical value around 9 A.
However, a single defined contact applied to all residue
types is a poor representationin a solvation potential. A
contact in a solvation potential should be defined as an
interaction which excludes solvent from the spacebetween
two residues. The difficulty in choosing a contact distance
is due to the variation in size among the twenty amino
acids. The Cp atomsof two large aromatic residuesmay
be 12 8, apart,yet water is excluded from the space
betweenthem due to the volume occupied by the side
chains. This samedistanceis not an appropriatecontact
definition for two alaninesbecausea water molecule could
easily passbetweenthe two side chains. A solvation
potential also requires a better estimation of the extent of
contact since a single definition would give the same
contact value to either glycine or tyrosine that is within the
cutoff distanceof a central residue. We addressthis
problem by using contact definitions which are a function
of the residuetypes involved and their distance apart.
For eachof the 210 possible amino acid pairs we derive
contact curves which representthe averagenumber of
atomic contacts(C,) observedin PDB structuresas a
function of the Cp distancebetweenthe specified pair of
residues. Thus, for structureswith simplified side chain
representationswe can provide an improved estimateof
interresidueinteractions and the extent of solvation. The
contact curves are obtained by tabulating all atomic
(heavy atom) contactsin over 1300PDB structuresfor
eachresiduepair. Our survey structuresare drawn from
the PDB Selectstructureswhich representprotein
sequenceswith no greaterthan 95% sequencehomology
(Hobohm, 1992). The proteins from the PDB Selectlist of
June 1997were screenedto eliminate structureswith chain
breaksand alternative coordinates;the remaining 1347
structuresbecameour survey set. To tabulate atomic
contactswe use the model of Colonna-Cesariand Sander
(1990) which considerstwo atomsfrom different side
chains to be in contact if the interatomic distanceis less
than or equal to 6.4 A. This distanceensuresexclusion of
a water molecule from betweenthe two atomsand is
derived by summing the van der Waals radii (2 x 1.8 A)
with the diameter of a water molecule (2.8 A). We use a
smooth sigmoidal curve with a maximum atomic contact
value of one when the atomsare less than 3.6 8, apart, and
a minimum value of zero when the distanceis greaterthan
6.4 A:
2
(d,, - U) (2d,, - 3 L + U)
ifLSd,,,,lU
a(&,, U, L) = (U- L)3
1 if d,,,, < L
1 0 ifd[,,, > U
where (II is the atomic contact betweenatoms1and m as a
function of the inter-atomic distanced,,, U is 6.4 A, and L
is 3.6 A. For each of the 210 amino acid pairs all atomic
contactsare tabulated from our survey structures. For each
pair we record the averagenumber of atomic contactsas a
function of the distancebetweenthe Cp carbonsof the
residuesinvolved, and then fit the data to a sigmoidal
curve. The contact curves for all 210 pairwise interactions
are then incorporated into the solvation potential. Figure 1
showsthe contact curve fit to the atomic contact data for
the proline-threonine pair. Cii is the averagenumber of
atomic contactsobservedwhen the Cp of the two residues
are the distancedii apart. This is one of the better fits with
a x2 of 0.65. In general,the x2 increaseswith residuesize.
The Solvation Potential
pairwise distancewe obtain the contact value Cii from the
appropriatecontact curve. All pairwise contact values are
then summedto give the total contact value for residue i.
The energy associatedwith residue i is the product of the
contact value and an energy parameterPk(i, for amino acid
type k. The potential has 21 adjustableparameters,20 of
which representthe solvation preferenceof the amino
acids and one parameter,P,, is usedin an overpacking
term. Equation (1) representsthe total energy of a given
sequencein a given conformation and is the sum of the
energy contribution from eachresidue in the protein plus
an overpacking term tabulated for eachresidue.
NN
E = ~,~,CijP~~i)+~P,Si (1)
i j i

In our solvation potential we utilize the approachof

Colonna-Cesariand Sander(1990) where a given amino N
acid type is assumedto have a fixed number of total
contactswhich are divided among solvent and protein 0 if c ij k(i), max
interactions. In this manner,solvation can be determined \j
implicitly by counting interresidue contacts. To assessthe Sj =
N N
extent of solvation for a given residue i, of amino acid type
k, we simply measurethe inter-($ distancesdii from the c ij - k(i), max if c ij > ck(i), max
specified residue to all others in the protein. For each i j

cij

1 -

10 15 20

dii (A>

Figure 1. Average number of atomic contactsCUobservedbetweenproline and

threonine as a function of the distancedii betweenCp carbons. The data points were
obtained from all proline-threonine interactions in 1347PDB structuresand were
binned in 0.25 8, increments. The dashedline representsthe sigmoid contact curve fit
to the data.

147
 60 80

c trp,total

Figure 2. Histogram of the observednumber of tryptophanswith a given total number of

atomic contacts(Cr,,,a3. The total atomic contactswere tabulated for 6303 tryptophans found
in 1347 PDB structuresand were separatedinto 100 bins. The maximum number of atomic
contactsobservedfor any tryptophan was 126.15. We use one standarddeviation below this
value as CQ,-, which is 106.69. During threading a steric penalty is incurred when C,V,- is
exceededfor any given tryptophan residue.

The summationsare performed over all N residues. The
overpacking term is the product of P, and Sj whereP, is
the overpacking parameterand Si is the excesscontact
value for residue i. Si is the amount by which the total
calculated inter-residuecontact value for residue i exceeds
a specified maximum value. Each amino acid type is
assigneda maximum contact value Ck(i),- which is
obtained from the distribution of total atomic contacts
observedin our survey structures. For eachamino acid of
type k found in our survey structureswe use our contact
definition curves to calculate the total number of
interresidue atomic contacts, The maximum contact
values for each amino acid type are calculated as follows:
for a given residue in a given structure the distanceto
every other residue in the structure is measuredas dij, and
for eachdti the atomic contact value is obtained from the
appropriatecontact curve. Atomic contact values are
summedfor the given residueproviding the total number
of atomic contactsper residue. A histogram for each
residue type provides a distribution of the number of
occurrencesof eachtotal contact value observedin the
survey. The contact value that is one standarddeviation
lower than the maximum observedis usedas Ck(i),mar
Figure 2 is the atomic contact distribution for tryptophan
obtainedfrom the 6303 tryptophans found in 1347 survey
structures. Taking one standarddeviation from the
maximum, we assignCrrp,- as 106.69.
Optimizing the Parameters
The adjustableparametersPk and P, are obtained through
an optimization procedurewhich ensuresthat for each
protein sequencein a training set, the PDB (native)
structure is the lowest in energy comparedto a very large
set of alternative conformations generatedby threading.
E alt - Enat m
Where Enatis the energy of the native sequencein the
native conformation, and Eal, is the energy of the native
sequencein an alternative conformation. The energiesare
calculated per equation (1). The constantm is an energy
margin set to 10. Each threading contributes an inequality,
and the parametersare obtained by solving the systemof
linear inequalities (Crippen, 1991;Maiorov & Crippen,

148
1992). The training procedurebegins with arbitrarily set
parameters,then a native sequenceis threadedthrough a
set of template structuresto produce the alternatives. As
each alternative is generated,we calculate the energy,
check compliance with equation (2) and continue to the
next threading if equation (2) is satisfied. Whenever a
violation of equation (2) is encountered,we add the current
inequality (constraint) to our problem, solve for the
parameters,then continue threading. The procedureis
repeatedfor each native and continues until equation (2) is
satisfied for every alternative structure producedfor every
native sequence. Structuresclassified as near-native are
excluded. Since we are satisfying inequalities, there can
be multiple solutions to a given problem. In this casewe
optimize the parametersby choosing the values which
minimize the sum of parameters.
It is possible to encounter an infeasible problem where
there is no solution to the set of linear inequalities. Care
must be taken when choosing the training set of proteins so
that we are not requiring the potential to satisfy a
constraint which is unreasonable. An example is
cytochrome c which hascovalently bound heme. Without
heme attached,apocytochromec is noncompactand does
not fold to its native structure (Dumont et al., 1994). We
found that including c-type cytochromesin the training set
results in an infeasible problem. However, since our
energy potential does not consider interactions with
prosthetic groups, metals, or ligands, we should not
demandthat the native structure of a protein dependenton
such interactions for native statestability be the lowest in
energy. In effect, we are calculating the energy of the apo
form of such proteins. Others have reportedpoor results
when testing potentials with proteins containing prosthetic
groups (Huang et al., 1995; Miyazawa and Jemigan,
1996). Here, we exclude theseproteins from our training
set.
Definition of Near-native, the Burial RMSD
Our goal is to construct a potential which can consistently
identify the native or a near-native structure for a given
protein sequenceout of a large set of alternatives. The
native structure is generally acceptedto be the crystal or
NMR structure in the PDB. The definition of near-native
is much more ambiguous. Similarity of two structuresis
conventionally measuredby the coordinate root-mean-
squaredeviation (RMSD) or the distanceRMSD (DME).
Thesemeasuresof similarity are visually appealingsince
they classify structuresthat look the sameas similar.
However, they are not satisfying when we consider
structureswhich are energetically near-native. Motion of
solvent exposedloops or hinge-like motion betweenlarge
149
protein domains do not necessarilyimply changesin free
energy yet may result in RMSD differences. It is desirable
to have a measureof structural similarity that is consistent
with our measureof energy. Since our energy potential is
a function of solvation, we wish to define near-native as
those structureswhich have a similar solvation profile as
the native. This definition allows for energetically similar
structuresto be classified as near-native despite coordinate
differences. We introduce the burial root-mean-square
deviation (BRMSD) as:
l/2
BRMSD = C1~Nl~tCi,a-Ci,~12
i 1
For a given sequencein two different conformations, N is
the number of residuesin the protein, C,, and C,, are the
total number of contactstabulated for residue i in
structuresa and b respectively using our contact
definitions.
To assessthe relationship of BRMSD to sequencelength
we selected18 PDB structureswhich ranged from 43 to
333 residuesin length. We threadedthe 18 sequences
through 121randomly selectedPDB structuresand
measuredthe BRMSD and DME for eachalternative in
referenceto the native structure. The averageBRMSD
showslittle dependenceon sequencelength (N) while the
averageDME has a clear dependence.Reva et al. (1998)
reportedthe averagecoordinate RMSD is proportional to
N3 for 142threadedproteins. The apparent
independenceof BRMSD on sequencelength is appealing
becauseone cutoff value may be suitable to define near-
native for proteins of various sequencelengths. To
determinewhether conventionally defined structural
homologuesfall within a given value of BRMSD we
comparedthe coordinate RMSD and BRMSD for 12
structural homologues(RMSD between0.4 and 2.4) of
protein 1351,turkey egg white lysozyme. Indeed, all
homologueshad a BRMSD below 10. Thus, we chosea
BRMSD of 10 as our near-native cutoff, and structures
with a BRMSD less than 10 relative to the native are
exempt from conformanceto equation (2) during training.
Initial Training and Evaluation
As a method of testing the solvation potential during
developmentand training we have initially used simple,
ungappedthreading for two reasons: 1) It remains the
minimal test that should be satisfied, i.e. if the native
structure cannot be consistently identified from a set of
ungappeddecoysthere is little hope that it will succeed
when faced with gappedstructures; 2) Numerous
ungappedthreading testshave appearedin the literature
and allow for comparison of results obtained with other
potential functions. The initial question which we sought
to addressis: Can the solvation potential, with a reasonable
amount of training, achieve comparableresults to those
previously established?
Initial training of the potential function using the training
set of Maiorov & Crippen (1992) followed by testing with
the test set of Miyazawa & Jernigan (1996) indicated that
small proteins and those with prosthetic groupsposedthe
greatestproblem. As mentioned previously, when c-type
cytochromeswere included in the training set the problem
becameinfeasible. In other words, no set of parameters
allowed every native structure to be the lowest in energy
for the correspondingnative sequencein the training set.
We found that by removing the cytochromesand two other
proteins (lhoe and 1hvp.A) from the RTS training set of
Maiorov & Crippen we were able to train the solvation
potential to correctly identify the training set native
structuresas the lowest in energy out of all alternatives.
We will refer to this set of natives as the initial training set
(ITS). The ability to achieve comparabletraining to
previous work is significant since we are now using only
2 1 adjustableparameterswhere Maiorov & Crippen had
used 112. It is desirable to usethe minimum number of
adjustableparametersto avoid overfitting which results in
a function well trained to identify a small number of
natives, but possibly incapable of accommodatingthe
general population of proteins.
The solvation potential and the parametersobtainedfrom
training with the ITS set were applied to the test set of
Miyazawa & Jernigan (1996). This test consistsof
threading 88 test sequencesthrough 189 template
structureswhich include the native structure for each
sequence.We did not use protein 2trx.A becauseseveral
residuesin the PDB structure have multiple conformers;
our test included the remaining 87 proteins. Excluding
proteins common to the training and test sets,our potential
function was successfulin identifying the native structure
for 73% of the test sequences.While not as successfulas
the 86% accuracy obtained by Miyazawa & Jemigan,it
should be noted that our training set consistedof only 27
proteins, and additional training promisesimproved
results. The results are less encouraging for proteins
shorter than 110 residues. We were only 50% accuratein
the Miyazawa & Jernigantest for theseproteins, while our
potential was able to correctly identify 83% of the proteins
greaterthan 110 residuesin length. It could be arguedthat
the poor performancewith small proteins is due to the
greater number of alternatives available from threading.
Smaller sequenceshave more decoy structuresproduced
by threading, resulting in a greaterchallenge. In the M & J
150
test a 54 residue sequencehas about 30,000 alternatives
while a 401 residueprotein has less than 1700 alternative
structuresavailable. Huber & Torda (1998) speculated
that their optimized potential may have provided better Z-
scoresfor larger proteins becauseof the fewer number of
alternativesavailable as comparedto smaller proteins.
They also noted that the common occurrenceof disulfide
bonds and post-translationalmodification in small proteins
may make their native structuresmore difficult to identify.
The former explanation suggeststhat the notable
performanceof contact potentials in ungappedthreading
testsof larger proteins may be simply due to the fewer
number of alternativesstructuresusedin the test. If we
use a very large number of alternative structures,can our
potential function still be trained to identify the correct
conformation for the native sequences?
Training with Extensive Threading
With the goal of training the solvation potential with a
much larger set of alternatives, we usedour entire set of
1347survey structuresas the threading template set. This
is over 10 times the number of template structuresused
with the ITS. Also, to focus on proteins greaterthan 110
amino acids in length, we createda new training set of
natives. To keep our training set within a computationally
manageablesize, we followed a few guidelines in selecting
the native proteins out of the nearly 8000 PDB structures
available. We startedwith our survey set which represents
a diverse set of proteins. Then, we limited candidatesto
those proteins between 110 and 500 residuesin length. It
is also desirableto use typical proteins, so we selected
those with averageamino acid composition. This was
done by classifying eachamino acid type as hydrophobic
(A,V,L,I,M,Y,W,F), polar (S,T,C,N,Q), charged
(K,R,E,D,H), or turn (G,P), then assessingthe composition
of eachprotein in our survey set and selecting those within
one standarddeviation of the mean. The last selection
criteria is the exclusion of proteins with prosthetic groups,
ligands, or a large number of metals. This reducedthe set
of candidatesdown to 69 proteins. The potential was
trained by first using a subsetof the candidatesas native
sequences,training by threading through our entire survey
set,then retaining those natives which contributed
constraintsto the problem. If an inequality (equation 2)
producedby threading a given native sequencethrough a
given structure is a solution boundary in the set of
inequalities, we consider it a constraint. We iteratively add
natives from the 69 candidatesuntil all are usedin at least
one round of training, while removing those which provide
no constraintsto the problem. Using this procedure,only
25 out of the 69 candidatesactually contributed constraints
to the problem. We will refer to this set of natives as the
extensive threading set (ETS).
Table 1. ETS Training Set.
The ETS along with the number of alternative structures
PDB length class alternatives
usedfor eachone are listed in Table 1. Note that the
solvation potential is able to identify the native structure as 1Paz 120 191478
the lowest in energy when comparedto nearly 135,000 ldro 122 188740
alternatives on average. Comparedwith the previous lbw4 125 184709
training set, where approximately 5000 alternativeswere leal 127 182080
usedfor a 170 residue sequence,we have provided a much 1351 129 179483
greaterchallenge. The optimized parametersare shown in 1arIl 129 179483
Table 2. A negative value indicates that interresidue lrsy 135 171958
contactsfor this amino acid type are favorable, while llcl 141 164647
amino acids with positive parameterstend to be exposedto llba 146 158727
solvent. It is reassuringthat the parametersare consistent 8ilb 146 158727
with generally acceptedhydrophobic classifications. We 2uce 148 156416
imposed an arbitrary boundary of -1000 5 Pk 5 1000 laak 150 154136
for eachparameter,and several parametersare at the lower 1891 164 139067
boundary. Our optimization calls for the solution which lsfe 165 138039
minimizes the sum of parameters;therefore, some llki 172 130967
parametershave a value of -1000 becausethey are not lhbq 177 127015
constrainedby existing inequalities of the problem. This lnfa 178 125082
condition suggeststhat the current solution spaceis large lwbc 183 120308
and further training can be accommodated. 1dsb.A 188 115663
lak2 220 88481
ljud 220 88481
lmml 251 68562
Testing the Parameters Obtained by Extensive Threading lapa 261 63048
lbtl 263 61982
The parametersshown in Table 2 were applied to the lagx 331 32779
Miyazawa & Jemigan test, and the results comparedto AVG <176> <134802>
those obtained in the initial training. The ETS results
exclude one protein that was common to both the ETS
training set and the M&J test set. Table 3 is a summaryof than 10. Apparently, for most proteins which fail the
results for the testsof the ITS and ETS parameters.The structure identification test, the problem is not due to
extensive threading parametersprovided slightly less decoyswith native-like folds. A representativeexample is
overall accuracy due mostly to a significant drop in ltpk.A, plasminogenactivator, with 88 residues. The
accuracy with the smaller proteins. Proteins greaterthan native structure ranked 22 out of over 23,000 alternatives.
110 residueswere correctly identified in about 82% of the The averageBRMSD of the 21 higher ranking alternatives
caseswith both setsof parameters.Excluding small is 16.51 which is about one standarddeviation from the
proteins from our extensive threading test set apparently meanBRMSD of the entire population of alternatives for
reduced the ability of the resulting potential to correctly 1tpk.A. The higher ranking decoysare not much more
identify small proteins. It is interesting to note that despite native-like than the generalpopulation of alternatives.
using two completely different training sets,of the 28 Further analysis of common characteristicsamong the
proteins which failed with ETS parameters,23 also failed higher ranking structuresmay reveal why they are
with the ITS parameters. This may reflect an intrinsic preferredover the native by the potential function.
weaknessof the solvation potential in its current form, Conversely,analysis of the native structureswhich fail
namely difficulty with small proteins. identification testsmay reveal common featuresamong
theseproteins which provide stability and are not currently
For the proteins which were incorrectly identified we accountedfor. In the M&J test 27 proteins are shorter than
examined those structureswhich ranked higher than the 110residuesin length (again excluding 2trx.A). Over half
native to find if they had native-like solvation profiles as of thesecontain disulfides. Of the 15 small, disulfide-
measuredby the BRMSD. For those natives which ranked bearing proteins 80% failed. Consideration of disulfides
lower than 50 we only checkedthe BRMSD of the top 50 and their contribution to native statestability may be
alternatives. Out of 28 failures only sevenhad a higher required.
ranking (lower energy) alternative with a BRMSD of less

151
 Table 2. ETS parametervalues for the 20 amino Table 3. Summaryof test results using the Miyazawa
acids and the steric penalty P,. Negative values & Jemiganthreading test. Figures representthe
percentageof structurescorrectly identified for the
reflect a favorable contact energy while ammo test sequences.Out of a total of 87 proteins, 60 have
acids with positive values tend to be exposed. a chain length greaterthan 110 residues. Proteins
common to training and test setswere not included in
ASN. 923.87 A the statistics.
ps 843.32
GLU 767.63 ITS ETS
GLN 492.88
ASP 411.67 overall 73.4 67.4
LYS 377.33
ARG 182.73 length > 110 82.5 81.4
PRO 84.72 Unfavorable Contact
SER 41.50 length < 110 50.0 37.0
HIS 29.54 -I

. . . . . . . . . . . 15.33
GLY ..... ................
THR -70.69 Given that fewer than 30 proteins were usedin each
-379.85 training set, the results indicate the approachis viable and
PHE -386.14 Favorable Contact promising. It is expectedthat an expandedtraining set of
MET -407.36 native proteins will provide additional constraints to the set
TYR -412.53 of inequalities and result in an improved potential. Current
LEU -580.94 parametersindicate that the existing solution spaceis large
VAL -893.91 and additional constraintscan be tolerated. Further
ILE -1000.00 training should be followed by an expandedrepertoire of
CYS -1000.00 teststo measurethe capability of the potential to identify
ALA -1000.00 v the native among decoysgeneratedby non-threading
methodssuch as off-lattice models and molecular
dynamics. Additional template structuresmay not be
beneficial to training. Further testing is required to
determineif training with our extensive set of alternative
structuresresults in a better potential comparedwith more
Conclusions limited training. Basedon the test results it is tempting to
conclude that our extensive threading produced a potential
We have constructeda potential function modeledon the no smarter than that obtained with our limited initial
predominant contribution to protein folding, namely training. However, differencesin the prediction ability of
solvation, while attempting to provide an improved the two setsof parametersmay not be apparentwith the
treatment of intramolecular interactions. Predefined current test. Despite the differencesbetweenthe two
contact definitions for all possible pairs of amino acids training sets,one would expect that parametersobtainedby
provide a method to assessthe extent of solvation using training with a much larger set of decoy structureswould
simplified side chain representations. The contact result in a more accuratepotential. Could it be that by
definitions are smooth and continuous as a function of increasingthe number of alternativesthrough threading we
interresidue distance. The potential function can be arejust adding more structuressimilar to those already
optimized so that the native structure for eachprotein used? Consideration of the quality of alternatives is
sequencein a training set is the lowest in energy compared warranted.
to an immenseset of alternative conformations. Testing
with the Miyazawa & Jemigan test showsthe potential
function to be greaterthan 80% accuratein identifying the
native structure for sequencesover 110 residuesin length, Acknowledgements
but shorter proteins are problematic. Results with two sets
of parametersobtained from independenttraining sets A.A.D. is a University of Michigan RackhamRegents
indicate numerouscommon failures. Small, disulfide- Fellow. This work is also supportedby the Michigan
bearing proteins are particularly difficult to identify, and Molecular Biophysics Training Program,National
consideration of disulfides may be required in the potential. Institutes of Health (GM08270).

152
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