Calculation and measurement of the dipole moment of small proteins: Use of protein data base

The dipole moments of small protein molecules were determined experimentally in order to validate the calculated dipole moments by previous investigators. We found that the agreements are satisfactory for some proteins. There are, however, many proteins for which the agreement is less than satisfactory. In order to find the cause of the disagreement, the dipole moments of these proteins were recalculated using the Brookhaven Protein Data Bank. We calculated the dipole moment due to fixed surface charges and the bond moments of all the carbonyl groups in main chain and side chains. The calculation consists of the mean moments and their mean square fluctuations. In addition, we investigated the effect of electrostatic interactions between charged sites for several proteins. These results show that incorporation of the interactions does not affect substantially the calculated dipole moments. The rms fluctuation of the dipole moment is found to be small but not negligible. In conclusion, recalculated dipole moments are in good agreement with the observed values. © 1993 John Wiley & Sons, Inc.     

Comparison of solvent-inaccessible cores of homologous proteins: definitions useful for protein modelling

The three-dimensional structures of 41 homologous proteins (belonging to eight families) were compared by pairwise superposition. A subset of 'core' residues was defined as those whose side chains have less than 7% of their surface exposed to solvent. This subset has significantly higher sequence identity and lower root mean square (RMS) alpha carbon separation than for all topologically equivalent residues in the structure, when members of a protein family are superposed. For such superpositions the relationship between RMS distance and percentage sequence identity of this subset of residues is similar to that for all equivalent residues, although some variation is observed between families of proteins which are predominantly beta sheet and those which are mainly alpha helix. The definition of a structurally more conserved core may be useful in model building proteins from an homologous family. The RMS differences of coordinates of structures of proteins with identical sequences are found to be related to the resolutions of the structures.     

A genetic algorithm that seeks native states of peptides and proteins.

We describe a computer algorithm to predict native structures of proteins and peptides from their primary sequences, their known native radii of gyration, and their known disulfide bonding patterns, starting from random conformations. Proteins are represented as simplified real-space main chains with single-bead side chains. Nonlocal interactions are taken from structural database-derived statistical potentials, as in an earlier treatment. Local interactions are taken from simulations of (phi, psi) energy surfaces for each amino acid generated using the Biosym Discover program. Conformational searching is done by a genetic algorithm-based method. Reasonable structures are obtained for melittin (a 26-mer), avian pancreatic polypeptide inhibitor (a 36-mer), crambin (a 46-mer), apamin (an 18-mer), tachyplesin (a 17-mer), C-peptide of ribonuclease A (a 13-mer), and four different designed helical peptides. A hydrogen bond interaction was tested and found to be generally unnecessary for helical peptides, but it helps fold some sheet regions in these structures. For the few longer chains we tested, the method appears not to converge. In those cases, it appears to recover native-like secondary structures, but gets incorrect tertiary folds.     

Protein contacts, inter-residue interactions and side-chain modelling

Three-dimensional structures of proteins are the support of their biological functions. Their folds are stabilized by contacts between residues. Inner protein contacts are generally described through direct atomic contacts, i.e. interactions between side-chain atoms, while contact prediction methods mainly used inter-Calpha distances. In this paper, we have analyzed the protein contacts on a recent high quality non-redundant databank using different criteria. First, we have studied the average number of contacts depending on the distance threshold to define a contact. Preferential contacts between types of amino acids have been highlighted. Detailed analyses have been done concerning the proximity of contacts in the sequence, the size of the proteins and fold classes. The strongest differences have been extracted, highlighting important residues. Then, we studied the influence of five different side-chain conformation prediction methods (SCWRL, IRECS, SCAP, SCATD and SCCOMP) on the distribution of contacts. The prediction rates of these different methods are quite similar. However, using a distance criterion between side chains, the results are quite different, e.g. SCAP predicts 50% more contacts than observed, unlike other methods that predict fewer contacts than observed. Contacts deduced are quite distinct from one method to another with at most 75% contacts in common. Moreover, distributions of amino acid preferential contacts present unexpected behaviours distinct from previously observed in the X-ray structures, especially at the surface of proteins. For instance, the interactions involving Tryptophan greatly decrease.     

Side chain burial and hydrophobic core packing in protein folding transition states

A critical step in the folding pathway of globular proteins is the formation of a tightly packed hydrophobic core. Several mutational studies have addressed the question of whether tight packing interactions are present during the rate-limiting step of folding. In some of these investigations, substituted side chains have been assumed to form native-like interactions in the transition state when the folding rates of mutant proteins correlate with their native-state stabilities. Alternatively, it has been argued that side chains participate in nonspecific hydrophobic collapse when the folding rates of mutant proteins correlate with side-chain hydrophobicity. In a reanalysis of published data, we have found that folding rates often correlate similarly well, or poorly, with both native-state stability and side-chain hydrophobicity, and it is therefore not possible to select an appropriate transition state model based on these one-parameter correlations. We show that this ambiguity can be resolved using a two-parameter model in which side chain burial and the formation of all other native-like interactions can occur asynchronously. Notably, the model agrees well with experimental data, even for positions where the one-parameter correlations are poor. We find that many side chains experience a previously unrecognized type of transition state environment in which specific, native-like interactions are formed, but hydrophobic burial dominates. Implications of these results to the design and analysis of protein folding studies are discussed.     

Four-body contact potentials derived from two protein datasets to discriminate native structures from decoys

Two-body inter-residue contact potentials for proteins have often been extracted and extensively used for threading. Here, we have developed a new scheme to derive four-body contact potentials as a way to consider protein interactions in a more cooperative model. We use several datasets of protein native structures to demonstrate that around 500 chains are sufficient to provide a good estimate of these four-body contact potentials by obtaining convergent threading results. We also have deliberately chosen two sets of protein native structures differing in resolution, one with all chains' resolution better than 1.5 A and the other with 94.2% of the structures having a resolution worse than 1.5 A to investigate whether potentials from well-refined protein datasets perform better in threading. However, potentials from well-refined proteins did not generate statistically significant better threading results. Our four-body contact potentials can discriminate well between native structures and partially unfolded or deliberately misfolded structures. Compared with another set of four-body contact potentials derived by using a Delaunay tessellation algorithm, our four-body contact potentials appear to offer a better characterization of the interactions between backbones and side chains and provide better threading results, somewhat complementary to those found using other potentials.     

Amino acid side-chain partition energies and distribution of residues in soluble proteins

Energies required to transfer amino acid side chains from water to less polar environments were calculated from results of several studies and compared with several statistical analyses of residue distributions in soluble proteins. An analysis that divides proteins into layers parallel with their surfaces is more informative than those that simply classify residues as exposed or buried. Most residues appear to be distributed as a function of the distance from the protein-water interface in a manner consistent with partition energies calculated from partitioning of amino acids between water and octanol phases and from solubilities of amino acids in water, ethanol, and methanol. Lys, Arg, Tyr, and Trp residues tend to concentrate near the water-protein interface where their apolar side-chain components are more buried than their polar side-chain components. Residue distributions calculated in this manner do not correlate well with side-chain solvation energies calculated from vapor pressures of side-chain analogs over a water phase. Results of statistical studies that classify residues as exposed to solvent or buried inside the protein interior appear to depend on the method used to classify residues. Data from some of these studies correlate better with solvation energies, but other data correlate better with partition energies. Most other statistical methods that have been used to evaluate effects of water on residue distributions yield results that correlate better with partition energies than with solvation energies.     

Methodological advancements for characterising protein side chains by NMR spectroscopy

The surface of proteins is covered by side chains of polar amino acids that are imperative for modulating protein functionality through the formation of noncovalent intermolecular interactions. However, despite their tremendous importance, the unique structures of protein side chains require tailored approaches for investigation by nuclear magnetic resonance spectroscopy and so have traditionally been understudied compared with the protein backbone. Here, we review substantial recent methodological advancements within nuclear magnetic resonance spectroscopy to address this issue. Specifically, we consider advancements that provide new insight into methyl-bearing side chains, show the potential of using non-natural amino acids and reveal the actions of charged side chains. Combined, the new methods promise unprecedented characterisations of side chains that will further elucidate protein function.     

Three-dimensional solution structure of apo-neocarzinostatin from Streptomyces carzinostaticus determined by NMR spectroscopy.

The three-dimensional solution structure of apo-neocarzinostatin has been resolved from nuclear magnetic resonance spectroscopy data. Up to 1034 constraints were used to generate an initial set of 45 structures using a distance geometry algorithm (DSPACE). From this set, ten structures were subjected to refinement by restrained energy minimization and molecular dynamics. The average atomic root mean square deviations between the final ten structures and the mean structure obtained by averaging their coordinates run from 0.085 nm for the best defined beta-sheet regions of the protein to 0.227 nm for the side chains of the most flexible loops. The solution structure of apo-neocarzinostatin is closely similar to that of the related proteins, macromomycin and actinoxanthin. It contains a seven-stranded antiparallel beta-barrel which forms, together with two external loops, a deep cavity that is the chromophore binding site. It is noteworthy that aromatic side chains extend into the binding cleft. They may be responsible for the stabilization of the holo-protein complex and for the chromophore specificity within the antitumoral family.     

Lipid interaction networks of peripheral membrane proteins revealed by data-driven micelle docking

Many signaling and trafficking proteins contain modular domains that bind reversibly to cellular membranes. The structural basis of the intermolecular interactions which mediate these membrane-targeting events remains elusive since protein-membrane complexes are not directly accessible to standard structural biology techniques. Here we report a fast protein-micelle docking methodology that yields three-dimensional model structures of proteins inserted into micelles, revealing energetically favorable orientations, convergent insertion angles, and an array of protein-lipid interactions at atomic resolution. The method is applied to two peripheral membrane proteins, the early endosome antigen 1 (EEA1) FYVE (a zinc finger domain found in the proteins Fab1, YOTB/ZK632.12, Vac1, and EEA1) and Vam7p phagocyte oxidase homology domains, which are revealed to form extensive networks of interactions with multiple phospholipid headgroups and acyl chains. The resulting structural models explain extensive published mutagenesis data and reveal novel binding determinants. The docking restraints used here were based on NMR data, but can be derived from any technique that detects insertion of protein residues into a membrane, and can be applied to virtually any peripheral membrane protein or membrane-like structure.     

Charge sequence coding in statistical modeling of unfolded proteins

Unfolded proteins recently attracted attention due to accumulation of experimental evidences for their significant role in different life processes. Modeling of electrostatic interactions (EI) in unfolded state of proteins is becoming increasingly important as well. In this paper, we stress on the importance of how the sequence of charged residues of a given protein is incorporated into the models for calculation of EI in the unfolded state. On the basis of the distributions of distances between titratable sites of charged residues calculated for polypeptide chains of various compositions, it was found that the distance distribution for a pair of residues, located close to each other along the sequence of a protein, depends on what residues constitute the pair in question. It was concluded that the consideration of these residue-specific distributions is essential for a statistical model to be accurate from the physical point of view. It was suggested that use of distance intervals in the spherical model of unfolded proteins accounts better for the charge sequence than the set of single distance values. This was illustrated by comparison of the pK values of the titratable groups of the unfolded N-terminal SH3 domain of the Drosophila protein drk to the available experimental data.     

Probing the energetic and structural role of amino acid/nucleobase cation-pi interactions in protein-ligand complexes

X-ray structures of proteins bound to ligand molecules containing a nucleic acid base were systematically searched for cation-pi interactions between the base and a positively charged or partially charged side chain group located above it, using geometric criteria. Such interactions were found in 38% of the complexes and are thus even more frequent than pi-pi stacking interactions. They are moreover well conserved in families of related proteins. The overwhelming majority of cation-pi contacts involve Ade bases, as these constitute by far the most frequent ligand building block; Arg-Ade is the most frequent cation-pi pair. Ab initio energy calculations at MP2 level were performed on all recorded pairs. Though cation-pi interactions involving the net positive charge carried by Arg or Lys side chains are the most favorable energetically, those involving the partial positive charge of Asn and Gln side chain amino groups (sometimes referred to as amino-pi interactions) are favorable too, owing to the electron correlation energy contribution. Chains of cation-pi interactions with a nucleobase bound simultaneously to two charged groups or a charged group sandwiched between two aromatic moieties are found in several complexes. The systematic association of these motifs with specific ligand molecules in unrelated protein sequences raises the question of their role in protein-ligand structure, stability, and recognition.     

The kinetics of side chain stabilization during protein folding

The rate of stabilization of side chains during protein folding has never been carefully studied. Recent developments in labeling proteins with (19)F-labeled amino acids coupled with real-time NMR measurements have allowed such measurements to be made. This paper describes the application of this method to the study of several proteins using 6-(19)F-tryptophan as the reporting group. It is found that these side chains adopt their final stable state at the last stages of the folding process and that the stabilization of side chains into their final conformation is a highly cooperative process. It is also possible to show the presence of intermediates in which the side chains are not correctly packed. The technique should be applicable to many systems.     

Evaluation of short-range interactions as secondary structure energies for protein fold and sequence recognition.

Short-range interactions for secondary structures of proteins are evaluated as potentials of mean force from the observed frequencies of secondary structures in known protein structures which are assumed to have an equilibrium distribution with the Boltzmann factor of secondary structure energies. A secondary conformation at each residue position in a protein is described by a tripeptide, including one nearest neighbor on each side. The secondary structure potentials are approximated as additive contributions from neighboring residues along the sequence. These are part of an empirical potential to provide a crude estimate of protein conformational energy at a residue level. Unlike previous works, interactions are decoupled into intrinsic potentials of residues, potentials of backbone-backbone interactions, and of side chain-backbone interactions. Also interactions are decoupled into one-body, two-body, and higher order interactions between peptide backbone and side chain and between backbones. These decouplings are essential to correctly evaluate the total secondary structure energy of a protein structure without overcounting interactions. Each interaction potential is evaluated separately by taking account of the correlation in the amino acid order of protein sequences. Interactions among side chains are neglected, because of the relatively limited number of protein structures. Proteins 1999;36:347-356. Published 1999 Wiley-Liss, Inc.     

Analysis of interactions of buried polar side chains in beta-proteins

It was shown in qualitative and quantitative analyses of polar side chains inaccessible for water molecules as well as their interactions in 100 globular beta-structural proteins that completely buried polar side chains are widespread in beta-proteins, their vast majority being involved in "side chain-side chain" or "side chain-main chain" interactions. The analysis of the occurrence of different "side chain-partner" pairs permitted us to demonstrate that such interactions are selective. The results were compared with similar data obtained previously for alpha-helical proteins.     

Analysis of Interactions of Buried Polar Side Chains in β-Proteins

Qualitative and quantitative analysis of polar side chains inaccessible to water molecules, as well as their interactions in 100 globular β-sheet proteins, was performed. It was shown that completely buried polar side chains are widespread in β-proteins, with their vast majority being involved in side chain-side chain or side chain-main chain interactions. An analysis of frequency of occurrence of different side chain-partner pairs demonstrated that these interactions are selective. The results were compared with similar data obtained earlier for α-helical proteins.     

Principles of membrane protein interactions with annular lipids deduced from aquaporin‐0 2D crystals

We have previously described the interactions of aquaporin‐0 (AQP0) with dimyristoyl phosphatidylcholine (DMPC) lipids. We have now determined the 2.5 Å structure of AQP0 in two‐dimensional (2D) crystals formed with Escherichia coli polar lipids (EPLs), which differ from DMPC both in headgroups and acyl chains. Comparison of the two structures shows that AQP0 does not adapt to the different length of the acyl chains in EPLs and that the distance between the phosphodiester groups in the two leaflets of the DMPC and EPL bilayers is almost identical. The EPL headgroups interact differently with AQP0 than do those of DMPC, but the acyl chains in the EPL and DMPC bilayers occupy similar positions. The interactions of annular lipids with membrane proteins seem to be driven by the propensity of the acyl chains to fill gaps in the protein surface. Interactions of the lipid headgroups may be responsible for the specific interactions found in tightly bound lipids but seem to have a negligible effect on interactions of generic annular lipids with membrane proteins.     

Analysis of side-chain rotamers in transmembrane proteins

We measured the frequency of side-chain rotamers in 14 alpha-helical and 16 beta-barrel membrane protein structures and found that the membrane environment considerably perturbs the rotamer frequencies compared to soluble proteins. Although there are limited experimental data, we found statistically significant changes in rotamer preferences depending on the residue environment. Rotamer distributions were influenced by whether the residues were lipid or protein facing, and whether the residues were found near the N- or C-terminus. Hydrogen-bonding interactions with the helical backbone perturbs the rotamer populations of Ser and His. Trp and Tyr favor side-chain conformations that allow their side chains to extend their polar atoms out of the membrane core, thereby aligning the side-chain polarity gradient with the polarity gradient of the membrane. Our results demonstrate how the membrane environment influences protein structures, providing information that will be useful in the structure prediction and design of transmembrane proteins.     

Membrane transport through α-heltical bundles. IV. Preliminary model building investigation of helix-helix interactions

Previously we made order of magnitude estimates that suggested the possibility of forming proton wires between facing α-helices joined by knobs-into-holes packing (Dunker, Marvin, Zaleske & Jones, 1976; Dunker & Marvin, 1978). Such structures may be a feature of membrane proteins. Since our original work, another type of packing, called ridges-into-grooves, has been identified as a way of meshing adjoining α-helices. Using precision (CPK) molecular models, we have investigated the possibility of forming proton wires: (1) in order to improve on our previous order of magnitude estimates, and (2) in order to evaluate the different kinds of packing interfaces for their ability to support stereochemically feasible proton wires.We found knobs-into-holes and ridges-into=grooves packing to support exactly the same proton wires. The positions of the side chains are determined more by the geometry of the hydrogen-bonding network rather than by the type of packing originally used to locate the appropriate residues. Thus, we suggest that a new name is needed for such packing, which we propose to call “H-bond packing”.In our earlier investigations we arbitrarily restricted our attention to proton wires parallel to the packing interface. By lifting this restriction, we found it possible to construct many additional types of wires. The model building exercises suggested that both parallel-to-the-interface and non-parallel-to-the-interface wires are feasible, except that the non-parallel wires are restricted in length depending on the angle with the interface, whereas the parallel wires apparently can be continued indefinitely. The various types of wires share local hydrogen bonding patterns and so could easily connect together.In our model building of several representative wires, we investigated the limitations with regard to type of and combinations of side chains. We also determined possible variations in the origins of such side chains on the helical backbones.These preliminary model building studies provide the basis for determining possible hydrogen bonding between the helical segments of membrane proteins. From these data we are formulating preliminary proposals for the helix-helix interactions of the bacteriorhodopsin molecule, which are to be presented in future paper.     

Identification of amino acids involved in protein structural uniqueness: implication for de novo protein design

Structural uniqueness is characteristic of native proteins and is essential to express their biological functions. The major factors that bring about the uniqueness are specific interactions between hydrophobic residues and their unique packing in the protein core. To find the origin of the uniqueness in their amino acid sequences, we analyzed the distribution of the side chain rotational isomers (rotamers) of hydrophobic amino acids in protein tertiary structures and derived deltaS(contact), the conformational-entropy changes of side chains by residue-residue contacts in each secondary structure. The deltaS(contact) values indicate distinct tendencies of the residue pairs to restrict side chain conformation by inter-residue contacts. Of the hydrophobic residues in alpha-helices, aliphatic residues (Leu, Val, Ile) strongly restrict the side chain conformations of each other. In beta-sheets, Met is most strongly restricted by contact with Ile, whereas Leu, Val and Ile are less affected by other residues in contact than those in alpha-helices. In designed and native protein variants, deltaS(contact) was found to correlate with the folding-unfolding cooperativity. Thus, it can be used as a specificity parameter for designing artificial proteins with a unique structure.