Disallowed Ramachandran conformations of amino acid residues in protein structures

An analysis of the nature and distribution of disallowed Ramachandran conformations of amino acid residues observed in high resolution protein crystal structures has been carried out. A data set consisting of 110 high resolution, non-homologous, protein crystal structures from the Brookhaven Protein Data Bank was examined. The data set consisted of a total of 18,708 non-Gly residues, which were characterized on the basis of their backbone dihedral angles (phi, psi). Residues falling outside the defined "broad allowed limits" on the Ramachandran map were chosen and the reported B-factor value of the alpha-carbon atom was used to further select well defined disallowed conformations. The conformations of the selected 66 disallowed residues clustered in distinct regions of the Ramachandran map indicating that specific phi, psi angle distortions are preferred under compulsions imposed by local constraints. The distribution of various amino acid residues in the disallowed residue data set showed a predominance of small polar/charged residues, with bulky hydrophobic residues being infrequent. As a further check, for all the 66 cases non-hydrogen van der Waals short contacts in the protein structures were evaluated and compared with the ideal "Ala-dipeptide" constructed using disallowed dihedral angle (phi, psi) values. The analysis reveals that short contacts are eliminated in most cases by local distortions of bond angles. An analysis of the conformation of the identified disallowed residues in related protein structures reveals instances of conservation of unusual stereochemistry.     

Predicting protein stability changes upon mutation using database-derived potentials: solvent accessibility determines the importance of local versus non-local interactions along the sequence

For 238 mutations of residues totally or partially buried in the protein core, we estimate the folding free energy changes upon mutation using database-derived potentials and correlate them with the experimentally measured ones. Several potentials are tested, representing different kinds of interactions. Local interactions along the chain are described by torsion potentials, based on propensities of amino acids to be associated with backbone torsion angle domains. Non-local interactions along the sequence are represented by distance potentials, derived from propensities of amino acid pairs or triplets to be at a given spatial distance. We find that for the set of totally buried residues, the best performing potential is a combination of a distance potential and a torsion potential weighted by a factor of 0.4; it yields a correlation coefficient between computed and measured changes in folding free energy of 0.80. For mutations of partially buried residues, the best potential is a combination of a torsion potential and a distance potential weighted by a factor of 0.7, and for the previously analysed mutations of solvent accessible residues, it is a torsion potential taken individually; the respective correlation coefficients reach 0.82 and 0.87. These results show that distance potentials, dominated by hydrophobic interactions, represent best the main interactions stabilizing the protein core, whereas torsion potentials, describing local interactions along the chain, represent best the interactions at the protein surface. The prediction accuracy reached by the distance potentials is, however, lower than that of the torsion potentials. A possible reason for this is that distance potentials would not describe correctly the effect on protein stability due to cavity formation upon mutating a large into a small amino acid. Last but not least, our results indicate that although local interactions, responsible for secondary structure formation, do not dominate in the protein core, they are not negligible for all that. They have a significant weight in the delicate balance between all the interactions that ensure protein stability.     

Protein structure prediction by threading. Why it works and why it does not

We developed a novel Monte Carlo threading algorithm which allows gaps and insertions both in the template structure and threaded sequence. The algorithm is able to find the optimal sequence-structure alignment and sample suboptimal alignments. Using our algorithm we performed sequence-structure alignments for a number of examples for three protein folds (ubiquitin, immunoglobulin and globin) using both "ideal" set of potentials (optimized to provide the best Z-score for a given protein) and more realistic knowledge-based potentials. Two physically different scenarios emerged. If a template structure is similar to the native one (within 2 A RMS), then (i) the optimal threading alignment is correct and robust with respect to deviations of the potential from the "ideal" one; (ii) suboptimal alignments are very similar to the optimal one; (iii) as Monte Carlo temperature decreases a sharp cooperative transition to the optimal alignment is observed. In contrast, if the template structure is only moderately close to the native structure (RMS greater than 3.5 A), then (i) the optimal alignment changes dramatically when an "ideal" potential is substituted by the real one; (ii) the structures of suboptimal alignments are very different from the optimal one, reducing the reliability of the alignment; (iii) the transition to the apparently optimal alignment is non-cooperative. In the intermediate cases when the RMS between the template and the native conformations is in the range between 2 A and 3.5 A, the success of threading alignment may depend on the quality of potentials used. These results are rationalized in terms of a threading free energy landscape. Possible ways to overcome the fundamental limitations of threading are discussed briefly.     

Age-related alterations in muscular endurance

BACKGROUND: Steric strain in protein three-dimensional structures is related to unfavorable inter-atomic interactions. The steric strain may be a result of packing or functional requirements, or may indicate an error in the coordinates of a structure. Detailed energy functions are, however, usually considered too noisy for error detection. RESULTS: After a short energy refinement, a full-atom, detailed energy function becomes a sensitive indicator of errors. The statistics of the energy distribution of amino acid residues in high-resolution crystal structures, represented by models with idealized covalent geometry, were calculated. The interaction energy of each residue with the whole protein structure and with the solvent was considered. Normalized deviations of amino acid residue energies from their average values were used for detecting energy-strained and, therefore, potentially incorrect fragments of a polypeptide chain. Protein three-dimensional structures of different origin (X-ray crystallography, NMR spectroscopy, theoretical models and deliberately misfolded decoys) were compared. Examples of the applications to loop and homology modeling are provided. CONCLUSIONS: Elevated levels of energy strain may point at a problematic fragment in a protein three-dimensional structure of either experimental or theoretical origin. The approach may be useful in model building and refinement, modeling by homology, protein design, folding calculations, and protein structure analysis.     

PROGEN: an automated modelling algorithm for the generation of complete protein structures from the alpha-carbon atomic coordinates

A modelling algorithm (PROGEN) for the generation of complete protein atomic coordinates from only the alpha-carbon coordinates is described. PROGEN utilizes an optimal geometry parameter (OGP) database for the positioning of atoms for each amino acid of the polypeptide model. The OGP database was established by examining the statistical correlations between 23 different intra-peptide and inter-peptide geometric parameters relative to the alpha-carbon distances for each amino acid in a library of 19 known proteins from the Brookhaven Protein Database (BPDB). The OGP files for specific amino acids and peptides were used to generate the atomic positions, with respect to alpha-carbons, for main-chain and side-chain atoms in the modelled structure. Refinement of the initial model was accomplished using energy minimization (EM) and molecular dynamics techniques. PROGEN was tested using 60 known proteins in the BPDB, representing a wide spectrum of primary and secondary structures. Comparison between PROGEN models and BPDB crystal reference structures gave r.m.s.d. values for peptide main-chain atoms between 0.29 and 0.76 A, with a grand average of 0.53 A for all 60 models. The r.m.s.d. for all non-hydrogen atoms ranged between 1.44 and 1.93 A for the 60 polypeptide models. PROGEN was also able to make the correct assignment of cis- or trans-proline configurations in the protein structures examined. PROGEN offers a fully automatic building and refinement procedure and requires no special or specific structural considerations for the protein to be modelled.     

Prediction of the three-dimensional structure of proteins using the electrostatic screening model and hierarchic condensation

We describe a method for predicting the three-dimensional (3-D) structure of proteins from their sequence alone. The method is based on the electrostatic screening model for the stability of the protein main-chain conformation. The free energy of a protein as a function of its conformation is obtained from the potentials of mean force analysis of high-resolution x-ray protein structures. The free energy function is simple and contains only 44 fitted coefficients. The minimization of the free energy is performed by the torsion space Monte Carlo procedure using the concept of hierarchic condensation. The Monte Carlo minimization procedure is applied to predict the secondary, super-secondary, and native 3-D structures of 12 proteins with 28-110 amino acids. The 3-D structures of the majority of local secondary and super-secondary structures are predicted accurately. This result suggests that control in forming the native-like local structure is distributed along the entire protein sequence. The native 3-D structure is predicted correctly for 3 of 12 proteins composed mainly from the alpha-helices. The method fails to predict the native 3-D structure of proteins with a predominantly beta secondary structure. We suggest that the hierarchic condensation is not an appropriate procedure for simulating the folding of proteins made up primarily from beta-strands. The method has been proved accurate in predicting the local secondary and super-secondary structures in the blind ab initio 3-D prediction experiment.     

Epithelioid angiomyolipoma of the kidney: a report of five cases with a prominent and diagnostically confusing epithelioid smooth muscle component

We suggest and test potentials for the modeling of protein structure on coarse lattices. The coarser the lattice, the more complete and faster is the exploration of the conformational space of a molecule. However, there are inevitable energy errors in lattice modeling caused by distortions in distances between interacting residues; the coarser the lattice, the larger are the energy errors. It is generally believed that an improvement in the accuracy of lattice modelling can be achieved only by reducing the lattice spacing. We reduce the errors on coarse lattices with lattice-adapted potentials. Two methods are used: in the first approach, 'lattice-derived' potentials are obtained directly from a database of lattice models of protein structure; in the second approach, we derive 'lattice-adjusted' potentials using our previously developed method of statistical adjustment of the 'off-lattice' energy functions for lattices. The derivation of off-lattice Calpha atom-based distance-dependent pairwise potentials has been reported previously. The accuracy of 'lattice-derived', 'lattice-adjusted' and 'off-lattice' potentials is estimated in threading tests. It is shown that 'lattice-derived' and 'lattice-adjusted' potentials give virtually the same accuracy and ensure reasonable protein fold recognition on the coarsest considered lattice (spacing 3.8 A), however, the 'off-lattice' potentials, which efficiently recognize off-lattice folds, do not work on this lattice, mainly because of the errors in short-range interactions between neighboring residues.     

New scoring schemes for protein fold recognition based on Voronoi contacts

MOTIVATION: The genome projects produce a wealth of protein sequences. Theoretical methods to predict possible structures and functions are needed for screening purposes, large-scale comparisons and in-depth analysis to identify worthwhile targets for further experimental research. Sequence-structure alignment is a basic tool for the identification of model folds for protein sequences and the construction of crude structural models. Empirical contact potentials (potentials of mean force) are used to optimize and evaluate such alignments. RESULTS: We propose new scoring schemes based on a contact definition derived from Voronoi decompositions of the three-dimensional coordinates of protein structures. We demonstrate that Voronoi potentials are superior to pure distance-based contact potentials with respect to recognition rate and significance for native folds. Moreover, the scoring scheme has the potential to provide a reasonable balance of detail and ion such that it is also useful for the recognition of distantly related (both homologous and non-homologous) proteins. This is demonstrated here on a set of structural alignments showing much better correspondence of native and model scores for the Voronoi potentials as compared to conventional distance-based potentials. AVAILABILITY: The potentials are made available via the program system ToPLign (URL: http://cartan.gmd.de/ToPLign.html). CONTACT: Ralf.Zimmer,Ralf.Thiele@gmd.de     

An efficient computational method for globally optimal threading

Computational recognition of native-like folds of an anonymous amino acid sequence from a protein fold database is considered to be a promising approach to the three-dimensional (3D) fold prediction of the amino acid sequence. We present a new method for protein fold recognition through optimally aligning an amino acid sequence and a protein fold template (protein threading). The fitness of aligning an amino acid sequence with a fold template is measured by (1) the singleton fitness, representing the compatibility of substituting one amino acid by another and the combined preference of secondary structure and solvent accessibility for a particular amino acid, (2) the pairwise interaction, representing the contact preference between a pair of amino acids, and (3) alignment gap penalties. Though a protein threading problem so defined is known to be NP-hard in the most general sense, our algorithm runs efficiently if we place a cutoff distance on the pairwise interactions, as many of the existing threading programs do. For an amino acid sequence of size n and a fold template of size m with M core secondary structures, the algorithm finds an optimal alignment in O (Mn1.5C + 1 + mnC + 1) time and O (MnC + 1) space, where C is a (small) nonnegative integer, determined by a particular mathematical property of the pairwise interactions. As a case study, we have demonstrated that C is less than or equal to 4 for about 75% of the 293 unique folds in our protein database, when pairwise interactions are restricted to amino acids < or = 7 A apart (measured between their beta carbon atoms). An approximation scheme is developed for fold templates with C > 4, when threading requires too much memory and time to be practical on a typical workstation.     

Solution structure of an artificial Fe8S8 ferredoxin: the D13C variant of Bacillus schlegelii Fe7S8 ferredoxin

The solution structure of the D13C variant of the thermostable Fe7S8 ferredoxin from Bacillus schlegelii has been determined by 1H-NMR spectroscopy in its oxidized form. In a variable-temperature NMR study the D13C variant was as thermostable (up to 90 degrees C) as the wild-type protein (WT). Seventy-five out of 77 amino acid residues and 81% of all theoretically expected proton resonances in the D13C Fe8S8 protein have been assigned. Its structure was determined through torsion angle dynamics calculations with the program DYANA, using 935 meaningful NOEs (from a total of 1251), hydrogen bond constraints, and NMR-derived dihedral angle constraints for the cluster-ligating cysteines. Afterwards, restrained energy minimization and restrained molecular dynamics were applied to each conformer of the family. The final family of 20 structures has RMSD values from the mean structure of 0.055 nm for the backbone atoms and of 0.099 nm for all heavy atoms. The overall folding of the WT is maintained in the mutant, except for the immediate vicinity of the new cysteine, which becomes much more similar to native Fe8S8 proteins. The two residues at positions 11 and 12, which constitute an insertion with respect to all known Fe8S8 proteins, assume a conformation that does not prevent the preceding and following residues from folding like in native Fe8S8 proteins. Clear evidence for the existence of two conformations involving almost half of the amino acid residues was found. The two conformations are structurally indistinguishable. Temperature-dependent NMR experiments show that one of them is thermodynamically more stable than the other.     

Traumatic myositis ossificans of the levator scapulae muscle

The Chou-Fasman method has been widely used for predicting protein secondary structure. It is based on knowledge of the potential of amino acid residues to form alpha-helical or beta-sheet regions in proteins. Our main interest in this study was to examine the reliability of these Chou-Fasman parameters. We calculated the Chou-Fasman parameters, with 95% confidence limits, of 144 non-homologous proteins consisting of 155 chains, and a total of 33 118 amino acid residues. All of the protein chains used were X-ray structures known at a resolution of at least 2.5 A. We compared the results of our calculations with those previously done by Chou and Fasman. Our results show that Chou and Fasman classified four amino acid residues wrongly in alpha-helical regions and one in a beta-sheet region. This is so, because the confidence limits we calculated did not include the values determined by Chou and Fasman. Moreover, the confidence limit calculations contradict most of the Chou-Fasman classification of amino acid residues.     

Easily searched protein folding potentials

In order to calculate the tertiary structure of a protein from its amino acid sequence, the thermodynamic approach requires a potential function of sequence and conformation that has its global minimum at the native conformation for many different proteins. Here we study the behavior of such functions for the simplest model system that still has the essential features of the protein folding problem, namely two-dimensional square lattice chain configurations involving two residue types. First we demonstrate a method for accurately recovering the given contact potential from only a knowledge of which sequences fold to which structures and what the non-native structures are. Second, we show how to derive from the same information more general potential functions having much better positive correlations between potential function value and conformational deviation from the native. These functions consequently permit faster and more reliable searches for the native conformation, given the native sequence. Furthermore, the method for finding such potentials is easily applied to more realistic protein models.     

Probing the structure and mechanism of Ras protein with an expanded genetic code

Mutations in Ras protein at positions Gly12 and Gly13 (phosphate-binding loop L1) and at positions Ala59, Gly60, and Gln61 (loop L4) are commonly associated with oncogenic activation. The structural and catalytic roles of these residues were probed with a series of unnatural amino acids that have unusual main chain conformations, hydrogen bonding abilities, and steric features. The properties of wild-type and transforming Ras proteins previously thought to be uniquely associated with the structure of a single amino acid at these positions were retained by mutants that contained a variety of unnatural amino acids. This expanded set of functional mutants provides new insight into the role of loop L4 residues in switch function and suggests that loop L1 may participate in the activation of Ras protein by effector molecules.     

Prediction and evaluation of side-chain conformations for protein backbone structures

A common approach to protein modeling is to propose a backbone structure based on homology or threading and then to attempt to build side chains onto this backbone. A fast algorithm using the simple criteria of atomic overlap and overall rotamer probability is proposed for this purpose. The method was first tested in the context of exhaustive searches of side chain configuration space in protein cores and was then applied to all side chains in 49 proteins of known structure, using simulated annealing to sample space. The latter procedure obtains the correct rotamer for 57% and the correct chi 1 value for 74% of the 6751 residues in the sample. When low-temperature Monte-Carlo simulations are initiated from the results of the simulated-annealing processes, consensus configurations are obtained which exhibit slightly more accurate predictions. The Monte-Carlo procedure also allows converged side chain entropies to be calculated for all residues. These prove to be accurate indicators of prediction reliability. For example, the correct rotamer is obtained for 79% and the correct chi 1 value is obtained for 84% of the half of the sample residues exhibiting the lowest entropies. Side chain entropy and predictability are nearly completely uncorrelated with solvent-accessible area. Some precedents for and implications of this observation are discussed.     

Partial amino acid sequence of gamma-46 gliadin

We have determined the partial amino acid sequence (207 amino acids) of gamma-46 gliadin isolated from wheat cultivar Hardi. The molecular mass of the protein (Mr) estimated by electrospray mass spectrometry is 35191.3. The number of cysteine residues in gamma-46 gliadin was determined as a mass difference of the protein before and after reduction and alkylation with 4-vinylpyridine. It was shown that the protein has no free SH-groups, and all cysteine residues are involved in the formation of four disulfide bonds. The partial structure of gamma-46 gliadin was determined by N-terminal sequencing and sequencing of tryptic and chymotryptic peptides. The tryptic peptides were obtained by enzymatic hydrolysis of the protein, which was preliminarily reduced and immobilized at free SH-groups on thiopropyl-Sepharose 6B. The chymotryptic peptides were isolated by limited digestion of the native protein. The positions of cysteine residues, as well as surrounding amino acid sequences, are conserved in gamma-46 gliadin; this is typical of gliadins.     

Physiological red blood cell kinetic model to explain the apparent discrepancy between adenosine breakdown inhibition and nucleoside transporter occupancy of draflazine

We present a fast method for finding optimal parameters for a low-resolution (threading) force field intended to distinguish correct from incorrect folds for a given protein sequence. In contrast to other methods, the parameterization uses information from >10(7) misfolded structures as well as a set of native sequence-structure pairs. In addition to testing the resulting force field's performance on the protein sequence threading problem, results are shown that characterize the number of parameters necessary for effective structure recognition.     

MONTY: a Monte Carlo approach to protein-DNA recognition

A Monte Carlo method is described for automated docking of proteins on DNA. The simulation program MONTY keeps the entire DNA and the protein backbone and core fixed while protein surface side-chains are allowed to rotate freely. The entire protein is rotated and translated by small random steps in order to find the best fit with the DNA. New configurations are accepted on basis of their Boltzmann probability. Protein-DNA interaction is represented by square well potentials for hydrogen bond and van der Waals interactions. The structure with the largest interaction energy encountered during the simulation is saved. The method is tested on complexes of the 434 Cro protein and its operator DNA where the protein is shifted up or down one or two base-pairs and is subsequently allowed to find back its native binding site. This protocol is performed for shifted complexes derived from the crystal structure, shifted complexes where the crystal structure DNA is replaced by standard B-DNA and shifted complexes where in addition the protein is replaced by protein from the uncomplexed crystal structure. In all three cases the six lowest energy structures correspond to complexes close to the native complex. The quality of sequence specific recognition diminishes, however, when the molecular surface complementarity between protein and DNA decreases.     

Lattice model simulations of polypeptide chain folding

Simulated annealing methods are applied to simple cubic lattice C alpha models of eight small monomeric globular proteins and their transition from a random chain to a low energy compact state is examined. The lowest energy structures are compared to their crystal forms using coordinate distance deviations, dRMS and RMS, and by distance contact maps. Analysis of the transition region indicates that, for this model, collapse begins with a rapid decline in radius of gyration followed continuously by chain repackings that lead to progressively lower values of chain energy. Chain repackings represent a highly cooperative interplay between the formation of local and non-local interactions. The components of this transition are characterized by rapid relaxation of shorter chain segments to form local contacts and slower relaxations of longer chain segments to form non-local contacts. Final structures obtained with this procedure contain many of the gross topologies of their native structures.     

Generation of monoclonal antibody specific to (1-->5)-alpha-L-arabinan

Monomeric bovine pancreatic RNase A has been transformed into a dimeric ribonuclease with antitumor activity (Di Donato, A., Cafaro, V. and D'Alessio, G. (1994) J. Biol. Chem. 269, 17394-17396). This was accomplished by replacing the residues located in the RNase chain at positions 19, 28, 31, and 32, with proline, leucine, and two cysteine residues, respectively, i.e. those present at identical positions in the subunit of bovine seminal RNase, a dimeric RNase of the pancreatic-type superfamily, endowed with a powerful antitumor action. However, as an antitumor agent this mutant dimeric RNase A is not as powerful as seminal RNase. We report here site-directed mutagenesis experiments which have led to the identification of two other amino acid residues, glycine 38 and 111, whose substitution in the polypeptide chain of the first generation dimeric mutant of RNase A, is capable of conferring to the mutein the full cytotoxic activity characteristic of native seminal RNase.