Long-term molecular dynamics simulation of copper plastocyanin in water

A long molecular dynamics simulation (1.1 ns) of fully hydrated plastocyanin has been performed and analysed to relate protein dynamics to structural elements and functional properties. The solvated structure is described in detail by the analysis of H-bond network. During all the simulation, the crystal H-bond network is maintained in the beta-sheet regions, while several H-bonds are broken or formed on the external surface of the protein. To evaluate whether such changes could be due to conformational rearrangements or to solvent competition, we have examined the average number of H-bonds between protein atoms and water molecules, and the root mean square deviations from crystal structure as a function of protein residues. Protein mobility and flexibility have been examined by positional and dihedral angle rms fluctuations. Finally, cross-correlation maps have revealed the existence of correlated motions among residues connected by hydrogen bonds.     

The aquaporin sidedness revisited

Aquaporins are transmembrane water channel proteins, which play important functions in the osmoregulation and water balance of micro-organisms, plants, and animal tissues. All aquaporins studied to date are thought to be tetrameric assemblies of four subunits each containing its own aqueous pore. Moreover, the subunits contain an internal sequence repeat forming two obversely symmetric hemichannels predicted to resemble an hour-glass. This unique arrangement of two highly related protein domains oriented at 180 degrees to each other poses a significant challenge in the determination of sidedness. Aquaporin Z (AqpZ) from Escherichia coli was reconstituted into highly ordered two-dimensional crystals. They were freeze-dried and metal-shadowed to establish the relationship between surface structure and underlying protein density by electron microscopy. The shadowing of some surfaces was prevented by protruding aggregates. Thus, images collected from freeze-dried crystals that exhibited both metal-coated and uncoated regions allowed surface relief reconstructions and projection maps to be obtained from the same crystal. Cross-correlation peak searches along lattices crossing metal-coated and uncoated regions allowed an unambiguous alignment of the surface reliefs to the underlying density maps. AqpZ topographs previously determined by AFM could then be aligned with projection maps of AqpZ, and finally with human erythrocyte aquaporin-1 (AQP1). Thereby features of the AqpZ topography could be interpreted by direct comparison to the 6 A three-dimensional structure of AQP1. We conclude that the sidedness we originally proposed for aquaporin density maps was inverted.     

Three-Dimensional Models of Immune Cell Surface Proteins and Identification of Binding Sites

The extracellular regions of many cell surface proteins of the immune system contain distinct domains that may be linked in many different ways and are often only loosely tethered to the transmembrane segment. In efforts to identify regions critical for binding, molecular models of these domains are used to select residues for mutagenesis and to map binding sites. Many immune cell surface proteins belong to protein superfamilies and display only limited sequence identity compared to proteins of known three-dimensional (3D) structure, often 30% or less. Therefore, detailed 3D structures are difficult to predict, and structure-based sequence analysis and model assessment are particularly important components of the model building process. In some cases, experimentally determined structures have made it possible to assess the accuracy of predictions, which illustrates the opportunities and shortcomings of the approach. Herein the model-based identification of binding sites in cell surface proteins is described and representative examples are discussed.     

Refined atomic model of the four-layer aggregate of the tobacco mosaic virus coat protein at 2.4-A resolution.

Previous x-ray studies (2.8-A resolution) on crystals of tobacco mosaic virus coat protein grown from solutions containing high salt have characterized the structure of the protein aggregate as a dimer of a bilayered cylindrical disk formed by 34 chemically identical subunits. We have determined the crystal structure of the disk aggregate at 2.4-A resolution using x-ray diffraction from crystals maintained at cryogenic temperatures. Two regions of interest have been extensively refined. First, residues of the low-radius loop region, which were not modeled previously, have been traced completely in our electron density maps. Similar to the structure observed in the virus, the right radial helix in each protomer ends around residue 87, after which the protein chain forms an extended chain that extends to the left radial helix. The left radial helix appears as a long alpha-helix with high temperature factors for the main-chain atoms in the inner portion. The side-chain atoms in this region (residues 90-110) are not visible in the electron density maps and are assumed to be disordered. Second, interactions between subunits in the symmetry-related central A pair have been determined. No direct protein-protein interactions are observed in the major overlap region between these subunits; all interactions are mediated by two layers of ordered solvent molecules. The current structure emphasizes the importance of water in biological macromolecular assemblies.     

Mapping of centromeric regions on the molecular linkage map of rice (Oryza sativa L.) using centromere-associated sequences

Genetic mapping of centromeres has been a challenge for plant geneticists. The objective of this study was to develop a new strategy for determining the locations of centromeric regions on genetic maps by mapping centromere-associated sequences, to make it possible to define the centromeric region of each chromosome as a single Mendelian locus on the molecular linkage map. Two DNA probes containing sequences specifically associated with the centromeres of grass species were used for genetic mapping. The centromere-associated sequences for all 12 rice chromosomes were mapped on the molecular map with either or both of the probes, and flanking molecular markers on one or both sides were localized 0 to 8 cM away. The map locations of the centromere-associated markers corresponded very well with the positions of centromeric regions determined previously using trisomic analyses for 11 of the 12 chromosomes. The precise mapping of the centromeric regions using these probes makes the molecular map a more complete and informative tool for genomic studies, which will facilitate studies of the structure and function of the rice centromeres. The simplicity of this technique, together with the fact that these probes are also associated with the centromeric regions in other grass species, may provide a general approach to the mapping of centromeric regions in the genomes of other cereal crops. Received: 8 July 1999 / Accepted: 19 November 1999     

Mapping the heparin-binding domain of human hepatic lipase

Human hepatic lipase (HL) is known to bind to the cell surface of hepatocytes and the sinusoidal endothelium of the liver. In each case, it appears that the enzyme remains associated with the cell surface through an ionic interaction with heparan sulfate proteoglycans. However, it remains unclear as to which residues are responsible for this critical function of the enzyme. In the present study, we have used a systematic approach to map the heparin-binding regions of human HL by utilizing peptide arrays spanning the complete sequence of the mature protein. Following probing with biotin-heparin, six peptides spanning residues 301-320 and 465-476 were identified as regions binding to heparin. Probing of an additional array containing these six parent peptides and a comprehensive series of mutant peptides identified two putative HL heparin-binding domains. The first was composed of residues R310, K312, K314, and R315 at the distal N-terminal domain and the second was composed of residues R473, K474, and R476 at the C-terminal end of the protein.     

Folding of beta/alpha-unit scrambled forms of S. cerevisiae triosephosphate isomerase: Evidence for autonomy of substructure formation and plasticity of hydrophobic and hydrogen bonding interactions in core of (beta/alpha)8-barrel

We show that residues at the interfaces of protein-protein complexes have higher side-chain energy than other surface residues. Eight different sets of protein complexes were analyzed. For each protein pair, the complex structure was used to identify the interface residues in the unbound monomer structures. Side-chain energy was calculated for each surface residue in the unbound monomer using our previously developed scoring function.1 The mean energy was calculated for the interface residues and the other surface residues. In 15 of the 16 monomers, the mean energy of the interface residues was higher than that of other surface residues. By decomposing the scoring function, we found that the energy term of the buried surface area of non-hydrogen-bonded hydrophilic atoms is the most important factor contributing to the high energy of the interface regions. In spite of lacking hydrophilic residues, the interface regions were found to be rich in buried non-hydrogen-bonded hydrophilic atoms. Although the calculation results could be affected by the inaccuracy of the scoring function, patch analysis of side-chain energy on the surface of an isolated protein may be helpful in identifying the possible protein-protein interface. A patch was defined as 20 residues surrounding the central residue on the protein surface, and patch energy was calculated as the mean value of the side-chain energy of all residues in the patch. In 12 of the studied monomers, the patch with the highest energy overlaps with the observed interface. The results are more remarkable when only three residues with the highest energy in a patch are averaged to derive the patch energy. All three highest-energy residues of the top energy patch belong to interfacial residues in four of the eight small protomers. We also found that the residue with the highest energy score on the surface of a small protomer is very possibly the key interaction residue.     

Structure-wise discrimination of cytosine,thymine, and uracil by proteins in terms of their nonbonded interactions

The molecular recognition and discrimination of very similar ligand moieties by proteins are important subjects in protein–ligand interaction studies. Specificity in the recognition of molecules is determined by the arrangement of protein and ligand atoms in space. The three pyrimidine bases, viz. cytosine, thymine, and uracil, are structurally similar, but the proteins that bind to them are able to discriminate them and form interactions. Since nonbonded interactions are responsible for molecular recognition processes in biological systems, our work attempts to understand some of the underlying principles of such recognition of pyrimidine molecular structures by proteins. The preferences of the amino acid residues to contact the pyrimidine bases in terms of nonbonded interactions; amino acid residue–ligand atom preferences; main chain and side chain atom contributions of amino acid residues; and solvent-accessible surface area of ligand atoms when forming complexes are analyzed. Our analysis shows that the amino acid residues, tyrosine and phenyl alanine, are highly involved in the pyrimidine interactions. Arginine prefers contacts with the cytosine base. The similarities and differences that exist between the interactions of the amino acid residues with each of the three pyrimidine base atoms in our analysis provide insights that can be exploited in designing specific inhibitors competitive to the ligands.     

Geometry of interaction of metal ions with sulfur-containing ligands in protein structures

An analysis of the geometry of binding of metal ions by cysteine and methionine residues in protein structures has been made by using the Protein Data Bank. Metal ions have a distinct model of binding to each of these residues, and this is independent of the nature of the metal center or the type of protein. Metal ions tend to approach the sulfur of Met roughly 38 degrees from the perpendicular to the plane through atoms C gamma-S delta-C epsilon. For Cys, the approach direction is such that the M...S gamma-C beta-C alpha torsional angle is about +/- 90 or 180 degrees. The side-chain conformation of the cysteine residue is affected by the presence of the metal ion; there is a shift from the g+ conformation toward g- and mainly t conformations. When two Cys residues at positions i-3 and i bind to the same metal center, there appears to be some restriction on the geometry of metal binding by the residue i; for such a residue chi 1 and M...S gamma-C beta-C alpha angles are likely to be around 60 degrees and 270 degrees, respectively. Met and Cys residues coordinating to a metal ion are usually from coil or turn regions of the protein structure.     

An anchored restriction-mapping approach applied to the genetic analysis of the Anopheles gambiae malaria vector complex 1

We introduce here a simple approach for rapidly determining restriction maps for a number of regions of a genome; this involves "anchoring" a map with a rare restriction site (in this case the seldom-cutting EagI) followed by partial digestion of a frequent-cutting enzyme (e.g., Sau 3A). We applied this technology to five species of the Anopheles gambiae complex. In a single Southern blot we obtained about a 15-kb restriction map each for the mtDNA, rRNA gene, and a scnDNA region for each of five species. Phylogenetic analyses of these regions yield trees at odds with the more traditional chromosome inversion-based trees. The value of the approach for systematic purposes is the ease with which several large, independent regions of the genome can be quickly assayed for molecular variation.      

On the characterization of energy networks of proteins

The construction of a realistic theoretical model of proteins is determinant for improving the computational simulations of their structural and functional aspects. Modeling proteins as a network of non-covalent connections between the atoms of amino acid residues has shown valuable insights into these macromolecules. The energy-related properties of protein structures are known to be very important in molecular dynamics. However, these same properties have been neglected when the protein structures are modeled as networks of atoms and amino acid residues. A new approach for the construction of protein models based on a network of atoms is presented. This method, based on interatomic interaction, takes into account the energy and geometric aspects of the protein structures that were not employed before, such as atomic occlusion inside the protein, the use of solvation, protein modeling and analysis, and the use of energy potentials to estimate the energies of interatomic non-covalent contacts. As a result, we achieved a more realistic network model of proteins. This model has the virtue of being more robust in face of different unknown variables that usually are arbitrarily estimated. We were able to determine the most connected residues of all the proteins studied, so that we are now in a better condition to study their structural role.     

Surface map comparison: studying function diversity of homologous proteins

A simplified protein surface cartography approach has been developed to assist in the analysis of surface features in homologous families, and thus to predict conservation or divergence of protein functions and protein-protein interaction patterns. A spherical approximation of protein surface was used, with a focus on charged and hydrophobic residues. The resulting surface map allows for qualitative analysis and comparison of surfaces of proteins, but can also be used to define a simple numerical measure of map similarity between two or more proteins. The latter was shown to be useful for function based classifications within large protein families.Surface map analysis was tested on several test cases: haemoglobins, death domains and TRAF domains. It was shown that surface map comparison allows a better function prediction than general sequence analysis methods and can reproduce known examples of functional variation within a divergent group of proteins. In another example, we predict novel, unexpected sets of common functional properties for seemingly distant members of a large group of divergent proteins. The method was also shown to be robust enough to allow using protein models from comparative modelling instead of experimental structures.     

Structure and inherent properties of the bacteriophage lambda head shell. VII. Molecular design of the form-determining major capsid protein

Some mutations in the major capsid protein (gpE) of lambda phage can alter the size and shape of the head shell or block the pathway of head maturation. Previous studies on the classification of such mutants showed that there are at least five functional sites on the gpE molecule. In this study, we determined the amino acid exchanges by DNA sequencing to elucidate the molecular design of the form-determining multifunctional protein gpE. In addition, we characterized the mutated gpE molecules by two-dimensional gel electrophoresis and studied suppression patterns of amber mutants at 43 amino acid residues. Those mutations map at 19 amino acid residues at 22 bases, which are located in three regions, 40 to 91, 222 to 246, and 284 to 324 of the 341 amino acid residues of gpE. These regions seem to be important in the activity of gpE, since amber mutations in these regions are suppressed on the average by less species of suppressors than those outside these regions. The mutations having different phenotypes are not segregated from each other, while some mutations having the same phenotype are separated far apart in the primary structure. This suggests that the functional sites were formed during evolution after the folding pattern of the ancestral gpE polypeptide chain had been established. Many of the mutations are located at serine, glycine and proline residues in predicted beta-turns.     

Identification of disulfide-containing peptides by performic acid oxidation and mass spectrometry

In addition to reducing the analysis time, the direct examination of proteolytic digests by fast atom bombardment mass spectrometry (FABMS) greatly extends the information that is available from peptide mapping experiments. Mass spectral data are particularly useful for identifying post-translationally modified peptides. For example, the molecular weight of a disulfide-containing peptide may be used to locate the disulfide bond in the protein from which the peptide was derived. This paper describes a new procedure, which is useful for identifying disulfide-bonded peptides. Peptides are treated with performic acid to modify certain residues and thereby cause a characteristic change in the peptide molecular weight. This change in molecular weight is determined by FABMS and used to help identify peptides. Results for a series of small peptides demonstrate that Cys, Met, and Trp are the only residues that undergo a change in molecular weight under the conditions used here. Furthermore, these changes in molecular weight are diagnostic for each of the residues. Cysteinyl-containing peptides are of particular interest, because their identification is essential for locating disulfide bonds. The molecular weight of a peptide increases by 48 mu for each cysteinyl residue present. This approach is used to identify peptides that contain both cysteinyl and cystinyl residues in the peptic digest of bovine insulin. The method is extended to the analysis of a tryptic digest of cyanogen bromide-treated ribonuclease A. A computer-assisted analysis procedure is used to demonstrate the specificity with which peptide molecular weight is related to specific segments of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Automated site-directed drug design: a general algorithm for knowledge acquisition about hydrogen-bonding regions at protein surfaces

This is the first of four papers that begin to explore the possibility of automated site-directed drug design. A general outline is given of the logical steps involved in approaching the problem. The starting point is the process of knowledge acquisition about the site. An algorithm is described here for the construction of a map of hydrogen-bonding regions at protein surfaces directly from the Brookhaven Protein Data Bank coordinates. Hydrogen-bonding atoms are located, intramolecular bonds are searched for, hydrogen-bonding atoms at the surface are found and hydrogen-bonding regions are computed at the accessible surface. A grid is placed within each region discovered and the probability of hydrogen bonding at each grid point is computed. The output of the program is a map of hydrogen-bonding regions displayed within a user-defined window. This information can be used as part of a knowledge base for the automatic construction of novel ligands to fit specified binding sites.     

A new method for mapping discontinuous antibody epitopes to reveal structural features of proteins.

Antibodies that bind to protein surfaces of interest can be used to report the three-dimensional structure of the protein as follows: Proteins are composed of linear polypeptide chains that fold together in complex spatial patterns to create the native protein structure. These folded structures form binding sites for antibodies. Antibody binding sites are typically "assembled" on the protein surface from segments that are far apart in the primary amino acid sequence of the target proteins. Short amino acid probe sequences that bind to the active region of each antibody can be used as witnesses to the antibody epitope surface and these probes can be efficiently selected from random sequence peptide libraries. This paper presents a new method to align these antibody epitopes to discontinuous regions of the one-dimensional amino acid sequence of a target protein. Such alignments of the epitopes indicate how segments of the protein sequence must be folded together in space and thus provide long-range constraints for solving the 3-D protein structure. This new antibody-based approach is applicable to the large fraction of proteins that are refractory to current approaches for structure determination and has the additional advantage of requiring very small amounts of the target protein. The binding site of an antibody is a surface, not just a continuous linear sequence, so the epitope mapping alignment problem is outside the scope of classical string alignment algorithms, such as Smith-Waterman. We formalize the alignment problem that is at the heart of this new approach, prove that the epitope mapping alignment problem is NP-complete, and give some initial results using a branch-and-bound algorithm to map two real-life cases. Initial results for two validation cases are presented for a graph-based protein surface neighbor mapping procedure that promises to provide additional spatial proximity information for the amino acid residues on the protein surface.     

Atomic mean-square displacements in proteins by molecular dynamics: a case for analysis of variance

Information on protein internal motions is usually obtained through the analysis of atomic mean-square displacements, which are a measure of variability of the atomic positions distribution functions. We report a statistical approach to analyze molecular dynamics data on these displacements that is based on probability distribution functions. Using a technique inspired by the analysis of variance, we compute unbiased, reliable mean-square displacements of the atoms and analyze them statistically. We applied this procedure to characterize protein thermostability by comparing the results for a thermophilic enzyme and a mesophilic homolog. In agreement with previous experimental observations, our analysis suggests that the proteins surface regions can play a role in the different thermal behavior.     

The 2.4-A crystal structure of Scapharca dimeric hemoglobin. Cooperativity based on directly communicating hemes at a novel subunit interface

The crystal structure of the cooperative dimeric hemoglobin from the arcid clam, Scapharca inaequivalvis, has been determined in the carbonmonoxy state. The phase problem was solved for reflections with Bragg spacings greater than 3 A using anomalous scattering from the porphyrin iron atoms measured at a single wavelength in combination with molecular averaging. The model built into this electron density map has been refined at 2.4 A resolution by means of stereochemically restrained least squares minimization to a conventional R-value of 0.156. The root mean square deviation from ideal bond lengths and angles are 0.013 A and 1.7 degrees, respectively. In addition to the 2336 hemoglobin atoms, 214 water molecules have been incorporated into the model. This structure reveals the details of an assemblage of two identical myoglobin-like subunits that is radically different from vertebrate hemoglobins. The subunit interface is formed by direct apposition of the E and F helices, whereas these surfaces are external in vertebrate hemoglobins. The interface has both hydrophobic and hydrophilic character. Two symmetrically related hydrophobic regions are formed between subunits. Six residues are involved in each of these regions that pack tightly enough to exclude water but have only a few atoms in close van der Waals contact. A number of ordered water molecules line the interface and form bridging hydrogen bonds between subunits. Four intersubunit ionic interactions are formed, two of which involve negatively charged propionate groups of the porphyrin. In contrast to cooperative vertebrate hemoglobins, a hydrogen bond network provides a direct route for communication between the two heme groups.     

Structural analysis of human and bovine alpha-fetoprotein by electron microscopy, image processing, and circular dichroism

The images of human and bovine alpha-fetoprotein molecules have been enhanced by combining dark-field electron microscopy with a laser-assisted optical system. This system filters out random background noise while permitting true averaged signal reconstruction of the molecule. A single averaged molecular image was digitized into a matrix, each pixel being assigned a gray scale level to produce a relative mass map for each molecule. These maps were interpreted from the alpha-helix, beta-form, and random coil of the purified proteins as determined by circular dichroism. Results showed that both molecules are "U shaped", apparently monomeric, with outside dimensions of approximately 80 A. Both molecules have asymmetrical structural features, notably three mass dense regions at both extremities and at the vertex of the molecules. Circular dichroism data suggest a high degree of similar stabilized alpha-helix and extensive beta-form in these regions. Mass map analysis of hAFP correlates with the subdomains organized by disulfide bridges.     

Dissection, residue conservation, and structural classification of protein-DNA interfaces

The basic DNA-binding modules of 128 protein-DNA interfaces have been analyzed. Although these are less planar, like the protein-protein interfaces, the protein-DNA interfaces can also be dissected into core regions in which all the fully-buried atoms are located, and rim regions having atoms with residual accessibilities. The sequence entropy of the core residues is smaller than those in the rim, indicating that the former are better conserved and possibly contribute more towards the binding free energy, as has been implicated in protein-protein interactions. On the protein side, 1014 A(2) of the surface is buried of which 63% belong to the core. There are some differences in the propensities of residues to occur in the core and the rim. In the DNA strands, the nucleotide(s) containing fully-buried atoms in all three components usually occupy central positions of the binding region. A new classification scheme for the interfaces has been introduced based on the composition of secondary structural elements of residues and the results compared with the conventional classification of DNA-binding proteins, as well as the protein class of the molecule. It appears that a common framework may be developed to understand both protein-protein and protein-DNA interactions.