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PubMed1990

Analysis of the structure of a common cold virus, human rhinovirus 14, refined at a resolution of 3.0 A.

Human rhinovirus 14 has a pseudo T = 3 icosahedral structure in which 60 copies of the three larger capsid proteins VP1, VP2 and VP3 are arranged in an icosahedral surface lattice, reminiscent of T = 3 viruses such as tomato bushy stunt virus and southern bean mosaic virus. The overall secondary and tertiary structures of VP1, VP2 and VP3 are very similar. The structure of human rhinovirus 14, which was refined at a resolution of 3.0 A [R = 0.16 for reflections with F greater than 3 sigma(F)], is here analyzed in detail. Quantitative analysis of the surface areas of contact (proportional to hydrophobic free energy of association) supports the previously assigned arrangement within the promoter, in which interactions between VP1 and VP3 predominate. Major contacts among VP1, VP2 and VP3 are between the beta-barrel moieties. VP4 is associated with the capsid interior by a distributed network of contacts with VP1, VP2 and VP3 within a promoter. As the virion assembly proceeds, the solvent-accessible surface area becomes increasingly hydrophilic in character. A mixed parallel and antiparallel seven-stranded sheet is composed of the beta C, beta H, beta E and beta F strands of VP3 in one pentamer and beta A1 and beta A2 of VP2 and the VP1 amino terminus in another pentamer. This association plays an essential role in holding pentamers together in the mature virion as this contact region includes more than half of the total short non-bonded contacts between pentamers. Contacts between protomers within pentamers are more extensive than the contacts between pentamers, accounting in part for the stability of pentamers. The previously identified immunogenic regions are correlated with high solvent accessibility, accessibility to large probes and also high thermal parameters. Surface residues in the canyon, the putative cellular receptor recognition site, have lower thermal parameters than other portions of the human rhinovirus 14 surface. Many of the water molecules in the ordered solvent model are located at subunit interfaces. A number of unusual crevices exist in the protein shell of human rhinovirus 14, including the hydrophobic pocket in VP1 which is the locus of binding for the WIN antiviral agents. These may be required for conformational flexibility during assembly and disassembly. The structures of the beta-barrels of human rhinovirus 14 VP1, VP2 and VP3 are compared with each other and with the southern bean mosaic virus coat protein.

Amino Acid Sequence↗

PubMed1996

Analysis of protein conformational characteristics related to thermostability.

The thermal stability of proteins was studied, 195 single amino acid residue replacements reported elsewhere being analysed for several protein conformational characteristics: type of residue replacement; conservative versus nonconservative substitution; replacement being in a homologous stretch of amino acid residues; change in hydrogen bond, van der Waals and secondary structure propensities; solvent-accessible versus inaccessible replacement; type of secondary structure involved in the substitution; the physico-chemical characteristics to which the thermostability enhancement can be attributed; and the relationship of the replacement site to the folding intermediates of the protein, when known. From the above analyses, some general rules arise which suggest where amino acid substitutions can be made to enhance protein thermostability: substitutions are conservative according to the Dayhoff matrix; mainly occur on conserved stretches of residues; preferentially occur on solvent-accessible residues; maintain or enhance the secondary structure propensity upon substitution; contribute to neutralize the dipole moment of the caps of helices and strands; and tend to increase the number of potential hydrogen bonding or van der Waals contacts or improve hydrophobic packing.

Amino Acid Sequence↗

PubMed2006

Average assignment method for predicting the stability of protein mutants.

Prediction of protein stability upon amino acid substitutions is an important problem in molecular biology and it will be helpful for designing stable mutants. In this work, we have analyzed the stability of protein mutants using three different data sets of 1791, 1396, and 2204 mutants, respectively, for thermal stability (DeltaTm), free energy change due to thermal (DeltaDeltaG), and denaturant denaturations (DeltaDeltaGH2O), obtained from the ProTherm database. We have classified the mutants into 380 possible substitutions and assigned the stability of each mutant using the information obtained with similar type of mutations. We observed that this assignment could distinguish the stabilizing and destabilizing mutants to an accuracy of 70-80% at different measures of stability. Further, we have classified the mutants based on secondary structure and solvent accessibility (ASA) and observed that the classification significantly improved the accuracy of prediction. The classification of mutants based on helix, strand, and coil distinguished the stabilizing/destabilizing mutants at an average accuracy of 82% and the correlation is 0.56; information about the location of residues at the interior, partially buried, and surface regions of a protein correctly identified the stabilizing/destabilizing residues at an average accuracy of 81% and the correlation is 0.59. The nine subclassifications based on three secondary structures and solvent accessibilities improved the accuracy of assigning stabilizing/destabilizing mutants to an accuracy of 84-89% for the three data sets. Further, the present method is able to predict the free energy change (DeltaDeltaG) upon mutations within a deviation of 0.64 kcal/mol. We suggest that this method could be used for predicting the stability of protein mutants.

Amino Acid Substitution↗

PubMed2002

Parameter optimized surfaces (POPS): analysis of key interactions and conformational changes in the ribosome.

We present a new method for the calculation of solvent accessible surface areas at the atomic and residue levels, which we call parameter optimized surfaces (POPS-A and POPS-R ). Atomic and residue areas (the latter simulated with a single sphere centered at the C(alpha)s atom for amino acids and at the P atom for nucleotides) have been optimized versus accurate all-atoms methods. We concentrated on an analytical formula for the approximation of solvent accessibilities. The formula is simple, easily derivable and fast to compute, therefore it is practical for use in molecular dynamics simulations as an approximation to the first solvation shell. The residue based approach POPS-R has been derived as a useful tool for the analysis of large macromolecular assemblies like the ribosome, and is especially suited for use in refinement of low resolution structures. The structures of the 70S, 50S and 30S ribosomes have been analyzed in detail and most of the interactions within the subunits and at their interfaces were clearly identified. Some interesting differences between 30S alone and within the 70S have been highlighted. Owing to the presence of the P-tRNA in the 70S ribosome, localized conformational rearrangements occur within the subunits, exposing Arg and Lys residues to negatively charged binding sites of P-tRNA. POPS-R also allows for estimates of the loss of free energy of solvation upon complex formation, particularly useful in designing new protein-RNA complexes and in suggesting more focused experimental work.

Models, Molecular↗

PubMed1999

Analysis of interactive packing of secondary structural elements in alpha/beta units in proteins.

An alpha-helix and a beta-strand are said to be interactively packed if at least one residue in each of the secondary structural elements loses 10% of its solvent accessible contact area on association with the other secondary structural element. An analysis of all such 5,975 nonidentical alpha/beta units in protein structures, defined at < or = 2.5 A resolution, shows that the interaxial distance between the alpha-helix and the beta-strand is linearly correlated with the residue-dependent function, log[(V/nda)/n-int], where V is the volume of amino acid residues in the packing interface, nda is the normalized difference in solvent accessible contact area of the residues in packed and unpacked secondary structural elements, and n-int is the number of residues in the packing interface. The beta-sheet unit (beta u), defined as a pair of adjacent parallel or antiparallel hydrogen-bonded beta-strands, packing with an alpha-helix shows a better correlation between the interaxial distance and log(V/nda) for the residues in the packing interface. This packing relationship is shown to be useful in the prediction of interaxial distances in alpha/beta units using the interacting residue information of equivalent alpha/beta units of homologous proteins. It is, therefore, of value in comparative modeling of protein structures.

Amino Acids↗

PubMed2006

Distill: a suite of web servers for the prediction of one-, two- and three-dimensional structural features of proteins.

BACKGROUND: We describe Distill, a suite of servers for the prediction of protein structural features: secondary structure; relative solvent accessibility; contact density; backbone structural motifs; residue contact maps at 6, 8 and 12 Angstrom; coarse protein topology. The servers are based on large-scale ensembles of recursive neural networks and trained on large, up-to-date, non-redundant subsets of the Protein Data Bank. Together with structural feature predictions, Distill includes a server for prediction of Calpha traces for short proteins (up to 200 amino acids). RESULTS: The servers are state-of-the-art, with secondary structure predicted correctly for nearly 80% of residues (currently the top performance on EVA), 2-class solvent accessibility nearly 80% correct, and contact maps exceeding 50% precision on the top non-diagonal contacts. A preliminary implementation of the predictor of protein Calpha traces featured among the top 20 Novel Fold predictors at the last CASP6 experiment as group Distill (ID 0348). The majority of the servers, including the Calpha trace predictor, now take into account homology information from the PDB, when available, resulting in greatly improved reliability. CONCLUSION: All predictions are freely available through a simple joint web interface and the results are returned by email. In a single submission the user can send protein sequences for a total of up to 32k residues to all or a selection of the servers. Distill is accessible at the address: http://distill.ucd.ie/distill/.

Algorithms↗

PubMed2001

FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties.

FUGUE, a program for recognizing distant homologues by sequence-structure comparison (http://www-cryst.bioc.cam.ac.uk/fugue/), has three key features. (1) Improved environment-specific substitution tables. Substitutions of an amino acid in a protein structure are constrained by its local structural environment, which can be defined in terms of secondary structure, solvent accessibility, and hydrogen bonding status. The environment-specific substitution tables have been derived from structural alignments in the HOMSTRAD database (http://www-cryst.bioc. cam.ac.uk/homstrad/). (2) Automatic selection of alignment algorithm with detailed structure-dependent gap penalties. FUGUE uses the global-local algorithm to align a sequence-structure pair when they greatly differ in length and uses the global algorithm in other cases. The gap penalty at each position of the structure is determined according to its solvent accessibility, its position relative to the secondary structure elements (SSEs) and the conservation of the SSEs. (3) Combined information from both multiple sequences and multiple structures. FUGUE is designed to align multiple sequences against multiple structures to enrich the conservation/variation information. We demonstrate that the combination of these three key features implemented in FUGUE improves both homology recognition performance and alignment accuracy.

Algorithms↗

PubMed1988

Surface amino acids as sites of temperature-sensitive folding mutations in the P22 tailspike protein.

Temperature-sensitive folding (tsf) mutations in the gene for the thermostable P22 tailspike interfere with the polypeptide chain folding and association pathway at restrictive temperature without altering the thermostability of the protein once correctly folded and assembled at permissive temperature. Though the native proteins matured at permissive temperature are biologically active, many of them display alterations in electrophoretic mobility. The native forms of 15 of these tsf mutant proteins have been purified and characterized. The purified proteins differed in electrophoretic mobility and isoelectric point from wild type but did not show evidence of major conformational alterations. The results suggest that the electrophoretic variations conferred by the 15 tsf amino acid substitutions are due to changes in the net charge at solvent-accessible sites in the native form of the mutant protein. During the maturation of the chains at restrictive temperature, these sites influence the conformation of intermediates in chain folding and association. The amino acid sequences at these sites resemble those found at turns in polypeptide chains. The isolation of tsf mutations requires that the mature structure of the tailspike accommodates the mutant amino acid substitution without loss of function. The solvent-accessible sites are probably at the surface of this structural protein. This would explain how bulky mutant substitutions, such as arginines for glycines, are accommodated in the native tailspike structure. Such sites, stabilizing intermediates in the folding pathway and located on the surface of the mature protein, probably represent a general class of conformational substrates for tsf mutations.

Amino Acids↗

PubMed1994

Resolution of structural changes associated with calcium activation of calmodulin using frequency domain fluorescence spectroscopy.

Structural changes associated with the calcium-dependent activation of wheat germ calmodulin (CaM) were assessed through measurements of steady-state and time-resolved changes in the fluorescence associated with (1) the unique tyrosine (Tyr139) located in calcium binding loop IV or (2) N-(1-pyrenyl)-maleimide (PM) or 4-(iodoacetamido)salicylic acid (IASA) covalently attached to Cys27 present in calcium binding loop I. These fluorophores permit the measurement of calcium-dependent changes in (i) the solvent accessibility and rotational dynamics associated with calcium binding loops I and IV and (ii) the hydrodynamic properties of the entire protein. Specific nitration of the unique tyrosine (Tyr139) in calcium binding loop IV permits the use of fluorescence resonance energy transfer to measure both the average spatial separation and distance heterogeneity between Cys27 and Tyr139, providing a direct measurement of the conformational flexibility of the central helix. Upon calcium binding, (i) the solvent accessibility and rotational dynamics of both PM and IASA (covalently bound to Cys27) and Tyr139 increase, (ii) overall protein rotational motion decreases, (iii) the average separation between the chromophores at Cys27 and nitrotyrosine 139 decreases, and (iv) the conformational flexibility associated with the central helix decreases. Therefore, upon calcium binding, the central helix becomes more extended and rigid, while the globular domains adopt a more open tertiary conformation that brings Cys27 and Tyr139 into closer proximity. This calcium-dependent structural change functions to expose the hydrophobic binding sites located within the globular domains, and to enhance the probability of binding target sequences through a reduction in conformational heterogeneity.

Calcium↗

PubMed2003

A fluorescence study of the structure and accessibility of plasmid DNA condensed with cationic gene delivery vehicles.

The cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane and dimethyldioctadecylammonium bromide, with or without the helper lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or cholesterol, and the cationic polymer polyethyleneimine, were compared for their ability to displace fluorescent dyes from DNA. Differences in displacement of the intercalating dyes ethidium bromide and ethidium homodimer correlate with their relative affinities with DNA, with the extent of ethidium homodimer displacement significantly less. Differences in ethidium homodimer and ethidium bromide displacement as a function of the ratio of polycation to DNA and the charge density of the polycation suggest a greater sensitivity of the former to topological changes in condensed DNA. Marked differences in the ability of these cationic delivery systems to displace the minor groove binding dyes 4',6-diamidino-2-phenylindole and Hoechst 33258 upon interaction with DNA are also apparent, with the majority of Hoechst 33258 remaining bound to DNA. Changes in the spectral properties of Hoechst 33258 were further used to characterize polycation-induced changes in solvent accessibility of the DNA minor groove. Taken together, these studies demonstrate differences in the interaction of various cationic lipids and polyethyleneimine in terms of regional displacement of dyes, polycation-induced structural changes in DNA, as well as polycation-mediated changes in solvent accessibility of the minor groove. The relevance of these studies to current models of the structure and assembly of polycation/DNA complexes are discussed.

Bisbenzimidazole↗

PubMed2000

Methylene as a possible universal footprinting reagent that will include hydrophobic surface areas: overview and feasibility: properties of diazirine as a precursor.

Methylene is one of, if not the, most reactive organic chemical known. It has a very low specificity, which makes it essentially useless for synthesis, but suggests a possible role in protein footprinting with special importance in labeling solvent accessible nonpolar areas, identifying ligand binding sites, and outlining interaction areas on protomers that form homo or hetero oligomers in cellular assemblies. The singlet species is easily and conveniently formed by photolysis of diazirine. The reactions of interest are insertion into C-H bonds and addition to multiple bonds, both forming strong covalent bonds and stable compounds. Reaction with proteins and peptides is reported even in aqueous solutions where the vast majority of the reagent is used up in forming methanol. Species containing up to 5 to 10 extra :CH2 groups are easily detected by electrospray mass spectroscopy. In a mixture of a 14 Kd protein and a noninteracting 1.7 Kd peptide, the distribution of mass peaks in the electrospray spectra was close to that expected from random modification of the estimated solvent accessible area for the two molecules. For analysis at the single residue level, quantitation at labeling levels of one 13CH2 group per 10 to 20 kDa of protein appears to be possible with isotope ratio mass spectroscopy. In the absence of reactive solvents, photolysis of diazirine produces oily polymeric species that contain one or two nitrogen atoms, but not more, and are water soluble.

Animals↗

PubMed2005

Mapping of the cyclodextrin cavity using the cylindrical coordinate system.

This paper describes the mapping of the inner cavity of cyclodextrin and the guest molecule using the cylindrical coordinate system. Each cavity structure of alpha-, beta-, and gamma-cyclodextrin was examined using the coordinate system, in which, the Z-axis passes through the molecular axis; a normal of the plane that consists of 6, 7, or 8 C(1) atoms of glucose units was obtained using the least-squares methods and that passes through the center of C(1) atoms. The radius rho is the distance from the Z-axis to the solvent-accessible surface of a cavity. The psi-axis is defined such that the nearest C(1) atom of the glucose unit to the Z-axis is its origin. The atomic character of the inner surface of the cavity was also obtained and plotted in the same coordinates. The guest molecule was plotted with the same coordinate system as cyclodextrin to see atoms with large rho distance of the guest molecule on the solvent-accessible surface map of cyclodextrin. It was confirmed from the map that cyclodextrin molecules have an ellipsoidal shape, have pseudo-n-hold symmetry (n=6 to alpha, 7 to beta, and 8 to gamma) in relation to their molecular axis, and guest molecules are located in relatively hydrophobic regions. It was also confirmed that the maps of this system give insights into the complementary characters of structures between the host and the guest by plotting the guest molecule in the same coordinate system.

Carbohydrate Conformation↗

PubMed2002

Domain organization of D-AKAP2 revealed by enhanced deuterium exchange-mass spectrometry (DXMS).

Dual specific A-kinase anchoring protein 2 (D-AKAP2) is a scaffold protein that coordinates cAMP-mediated signaling complexes by binding to type I and type II protein kinase A (PKA). While information is unfolding regarding specific binding motifs, very little is known about the overall structure and dynamics of these scaffold proteins. We have used deuterium exchange-mass spectrometry (DXMS) and limited proteolysis to probe the folded regions of D-AKAP2, providing for the first time insight into the intra-domain dynamics of a scaffold protein. Deuterium on-exchange revealed two regions of low deuterium exchange that were surrounded by regions of high exchange, suggestive of two distinctly folded regions, flanked by disordered or solvent accessible regions. Similar folded regions were detected by limited proteolysis. The first folded region contained a putative regulator of G-protein signaling (RGS) domain. A structural model of the RGS domain revealed that the more deuterated regions mapped onto loops and turns, whereas less deuterated regions mapped onto alpha-helices, consistent with this region folding into an RGS domain. The second folded region contained a highly protected PKA binding site and a more solvent-accessible PDZ binding motif, which may serve as a potential targeting domain for D-AKAP2. DXMS has verified the multi-domain architecture of D-AKAP2 implied by sequence homology and has provided unique insight into the accessibility of the PKA binding site.

A Kinase Anchor Proteins↗

PubMed2000

Substitutions of surface amino acid residues of cutinase probed by aqueous two-phase partitioning.

The surface properties of a protein are often crucial for recognition and interaction with other molecules. Important functional residues can be identified by mutational analysis. There is a need for rapid methods to study protein surfaces and surface changes due to mutations. Partitioning in aqueous two-phase systems has the potential to be used in this respect since protein partitioning depends on the surface properties of the protein. The influence of surface-exposed amino acid residues in protein partitioning has been studied with cutinase variants, which differed in one or several amino acid residues as a result of site-directed mutagenesis. The solvent accessibility of the mutated residues was determined with a computer program, Graphical Representation and Analysis of Surface Properties. The aqueous two-phase system was composed of dextran and a random copolymer of ethylene oxide and propylene oxide. It was shown, for the first time, to what extent surface-exposed amino acid residues influence the partition coefficient in an aqueous two-phase system. The effect on partitioning could be described only taking into account solvent accessibility and type of residue substitution. The results demonstrate that the system can be used to detect conformational changes in mutant proteins since the expected effect on partitioning due to a mutation can be calculated. The aqueous two-phase system used here does indeed provide a rapid and convenient method to study protein surfaces and slight surface changes due to mutations.

Amino Acid Substitution↗

PubMed2000

Protein gamma-radiolysis in frozen solutions is a macromolecular surface phenomenon: fragmentation of lysozyme, citrate synthase and alpha-lactalbumin in native or denatured states.

PURPOSE: To test whether radiolysis-induced fragmentation in frozen aqueous protein solution is dependent on solvent access to the surface of the protein or to the molecular mass of the polypeptide chain. MATERIALS AND METHODS: 60Co gamma-irradiation of three proteins at -78 degrees C: lysozyme, citrate synthase and alpha-lactalbumin in their native state, with or without bound substrate, or denatured (random coil in urea/acid-denatured state). RESULTS: By SDS-polyacrylamide gel electrophoresis/analysis of the protein-fragmentation process, it was found that for a given protein D37 values (dose to decrease the measured amount of protein, with an unaltered polypeptidic chain, to 37% of the initial amount) varied according to the state of the protein. D37 for denatured proteins was always much smaller than for native states, indicating a greater susceptibility to fragmentation. In urea, contrary to the native state, no well-defined fragments were observed. Radiolysis decay constants (K= 1/D37) increased with solvent-accessible surface area of these proteins estimated from their radii of gyration in the various states. This is shown also in previous data on native or SDS-denatured proteins. Denatured proteins which have a large surface area exposed to the solvent compared with native ones are more fragmented at equal doses. CONCLUSIONS: It is concluded that D37 is directly related to the exposed surface area and not to the molecular mass of the polypeptide chain.

Animals↗

PubMed2003

Bacillus subtilis SecA ATPase exists as an antiparallel dimer in solution.

SecA ATPase promotes the biogenesis of membrane and secretory proteins into and across the cytoplasmic membrane of Eubacteria. SecA binds to translocon component SecYE and substrate proteins and undergoes ATP-dependent conformational cycles that are coupled to the stepwise translocation of proteins. Our recent crystal structure of B. subtilis SecA [Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018-2026] showed two different dimer interactions in the lattice which both buried significant solvent-accessible surface area in their interface and could potentially be responsible for formation of the physiological dimer in solution. In this paper, we utilize fluorescence resonance energy transfer methodology with genetically engineered SecA proteins containing unique pairs of tryptophan and fluorophore-labeled cysteine residues to determine the oligomeric structure of SecA protein in solution. Our results show that of the two dimers interactions observed in the crystal structure, SecA forms an antiparallel dimer in solution that maximizes the buried solvent-accessible surface area and intermolecular contacts. At the submicromolar protein concentrations used in the fluorescence experiments, we saw no evidence for the formation of higher-order oligomers of SecA based on either the alternative dimer or the 3(1) helical fiber observed in the crystal lattice. Our studies are consistent with previous ones demonstrating the existence of a dimerization determinant within the C-domain of SecA as well as those documenting the interaction of N- and C-domains of SecA. Our results also provide a valuable starting point for a determination of whether the subunit status of SecA changes during the protein translocation as well as studies designed to elucidate the conformational dynamics of this multidomain protein during its translocation cycle.

Adenosine Triphosphatases↗

PubMed2005

Secondary reactions and strategies to improve quantitative protein footprinting.

Hydroxyl radical-mediated footprinting permits detailed examination of structure and dynamic processes of proteins and large biological assemblies, as changes in the rate of reaction of radicals with target peptides are governed by changes in the solvent accessibility of the side-chain probe residues. The precise and accurate determination of peptide reaction rates is essential to successfully probing protein structure using footprinting. In this study, we specifically examine the magnitude and mechanisms of secondary oxidation occurring after radiolytic exposure and prior to mass spectrometric analysis. Secondary oxidation results from hydrogen peroxide and other oxidative species generated during radiolysis, significantly impacting the oxidation of Met and Cys but not aromatic or other reactive residues. Secondary oxidation of Met with formation of sulfoxide degrades data reproducibility and inflates the perceived solvent accessibility of Met-containing peptides. It can be suppressed by adding trace amounts of catalase or millimolar Met-NH2 (or Met-OH) buffer immediately after irradiation; this leads to greatly improved adherence to first-order kinetics and more precise observed oxidation rates. The strategy is shown to suppress secondary oxidation in model peptides and improve data quality in examining the reactivity of peptides within the Arp2/3 protein complex. Cysteine is also subject to secondary oxidation generating disulfide as the principal product. The disulfides can be reduced before mass spectrometric analysis by reducing agents such as TCEP, while methionine sulfoxide is refractory to reduction by this reagent under typical reducing conditions.

Cysteine↗

PubMed2005

Assessing methods for identifying pair-wise atomic contacts across binding interfaces.

An essential step in understanding the molecular basis of protein-protein interactions is the accurate identification of inter-protein contacts. We evaluate a number of common methods used in analyzing protein-protein interfaces: a Voronoi polyhedra-based approach, changes in solvent accessible surface area (DeltaSASA) and various radial cutoffs (closest atom, Cbeta, and centroid). First, we compared the Voronoi polyhedra-based analysis to the DeltaSASA and show that using Voronoi polyhedra finds knob-in-hole contacts. To assess the accuracy between the Voronoi polyhedra-based approach and the various radial cutoff methods, two sets of data were used: a small set of 75 experimental mutants and a larger one of 592 structures of protein-protein interfaces. In an assessment using the small set, the Voronoi polyhedra-based methods, a solvent accessible surface area method, and the closest atom radial method identified 100% of the direct contacts defined by mutagenesis data, but only the Voronoi polyhedra-based method found no false positives. The other radial methods were not able to find all of the direct contacts even using a cutoff of 9A. With the larger set of structures, we compared the overall number contacts using the Voronoi polyhedra-based method as a standard. All the radial methods using a 6-A cutoff identified more interactions, but these putative contacts included many false positives as well as missed many false negatives. While radial cutoffs are quicker to calculate as well as to implement, this result highlights why radial cutoff methods do not have the proper resolution to detail the non-homogeneous packing within protein interfaces, and suggests an inappropriate bias in pair-wise contact potentials. Of the radial cutoff methods, using the closest atom approach exhibits the best approximation to the more intensive Voronoi calculation. Our version of the Voronoi polyhedra-based method QContacts is available at .

Databases, Protein↗