Marvin Edelman

Automated analysis of interatomic contacts in proteins.

MOTIVATION New software has been designed to assist the molecular biologist in understanding the structural consequences of modifying a ligand and/or protein. RESULTS Tools are described for the analysis of ligand-protein contacts (LPC software) and contacts of structural units (CSU software) such as helices, sheets, strands and residues. Our approach is based on a detailed analysis of interatomic contacts and interface complementarity. For any ligand or structural unit, these software automatically: (i) calculate the solvent-accessible surface of every atom; (ii) determine the contacting residues and type of interaction they undergo (hydrophobic-hydrophobic, aromatic-aromatic, etc.); (iii) indicate all putative hydrogen bonds. LPC software further predicts changes in binding strength following chemical modification of the ligand. AVAILABILITY Both LPC and CSU can be accessed through the PDB and are integrated in the 3DB Atlas page of all PDB files. For any given file, the tools can also be accessed at http://www.pdb.bnl. gov/pdb-bin/lpc?PDB_ID= and http://www.pdb.bnl. gov/pdb-bin/csu?PDB_ID= with the four-letter PDB code added at the end in each case. Finally, LPC and CSU can be accessed at: http://sgedg.weizmann.ac.il/lpc and http://sgedg.weizmann.ac.il/csu.

Side-Chain Flexibility in Proteins Upon Ligand Binding

Ligand binding may involve a wide range of structural changes in the receptor protein, from hinge movement of entire domains to small side‐chain rearrangements in the binding pocket residues. The analysis of side chain flexibility gives insights valuable to improve docking algorithms and can provide an index of amino‐acid side‐chain flexibility potentially useful in molecular biology and protein engineering studies. In this study we analyzed side‐chain rearrangements upon ligand binding. We constructed two non‐redundant databases (980 and 353 entries) of “paired” protein structures in complexed (holo‐protein) and uncomplexed (apo‐protein) forms from the PDB macromolecular structural database. The number and identity of binding pocket residues that undergo side‐chain conformational changes were determined. We show that, in general, only a small number of residues in the pocket undergo such changes (e.g., ∼85% of cases show changes in three residues or less). The flexibility scale has the following order: Lys > Arg, Gln, Met > Glu, Ile, Leu > Asn, Thr, Val, Tyr, Ser, His, Asp > Cys, Trp, Phe; thus, Lys side chains in binding pockets flex 25 times more often then do the Phe side chains. Normalizing for the number of flexible dihedral bonds in each amino acid attenuates the scale somewhat, however, the clear trend of large, polar amino acids being more flexible in the pocket than aromatic ones remains. We found no correlation between backbone movement of a residue upon ligand binding and the flexibility of its side chain. These results are relevant to 1. Reduction of search space in docking algorithms by inclusion of side‐chain flexibility for a limited number of binding pocket residues; and 2. Utilization of the amino acid flexibility scale in protein engineering studies to alter the flexibility of binding pockets. Proteins 2000;39:261–268.

Identification of a primary in vivo degradation product of the rapidly-turning-over 32 kd protein of photosystem II

The 32 kd photosystem II protein of plant chloroplasts is rapidly turned over in the light. The initial events in the degradation of the 32 kd protein were studied. A 23.5 kd breakdown product was identified in Spirodela oligorrhiza membranes using immunological analysis. The 23.5 kd polypeptide was shown to be derived from the amino‐terminal portion of the 32 kd protein using partial proteolytic fingerprinting. An in vivo precursor–product relationship between the 32 kd protein and the 23.5 kd polypeptide was kinetically demonstrated by radiolabeling and pulse‐chase experiments. The cleavage site yielding the 23.5 kd polypeptide was localized to a functionally active region (between helices IV and V) of the 32 kd protein. We propose that an alpha‐helix‐destabilizing ‘degradation’ sequence, bordered by arginine residues 225 and 238, is involved in the formation of the 23.5 kd polypeptide.

Studies of chloroplast development in Euglena: XII. Two types of satellite DNA

Three species of DNA from wild-type cells of Euglena gracilis var. bacillaris are described. Main band DNA (density = 1·707 g/cm3) most probably is assignable to the nucleus and is found in all strains examined. The satellite region (density = 1·688 g/cm3) previously described ( Leff, Mandel, Epstein & Schiff, 1963 ) has now been resolved into two DNA satellites, Sc (density = 1·686 g/cm3) and Sx (density = 1·691 g/cm3), both of which are present in wild-type cells, Sc is found in enriched quantities in the chloroplast fraction of green cells and is found in light-grown and dark-grown wild-type cells and in all mutants capable of forming at least a partial chloroplast, but is absent from all mutants incapable of plastid formation. Conversion of strains capable of chloroplast formation to strains incapable of this by means of ultraviolet treatment brings about a loss of Sc. This is consistent with ScDNA being a constituent of the chloroplasts. Sx is found in wild-type cells and in all mutants examined, whether capable or incapable of forming chloroplasts. On cell fractionation, Sx is absent from the chloroplast fraction and is found in the small-particle fraction of the cells along with mitochondrial cytochromes. From density calculations, main band DNA contains 48% guanine+cytosine : 52% adenine+thymine; Sx contains 31% guanine+cytosine: 69% adenine+thymine; and Sc contains 26% guanine+cytosine : 74% adenine+thymine. All three DNA species appear to be double-stranded.

D1-protein dynamics in photosystem II: the lingering enigma

The D1/D2 heterodimer core is the heart of the photosystem II reaction center. A characteristic feature of this heterodimer is the differentially rapid, light-dependent degradation of the D1 protein. The D1 protein is possibly the most researched photosynthetic polypeptide, with aspects of structure–function, gene, messenger and protein regulation, electron transport, reactive oxygen species, photoinhibition, herbicide binding, stromal–granal translocations, reversible phosphorylation, and specific proteases, all under intensive investigation more than three decades after the protein’s debut in the literature. This review will touch on some treaded areas of D1 research that have, so far, defied clear resolution, as well as cutting edge research on mechanisms and consequences of D1 protein degradation.

Control of psbA gene expression: in mature Spirodela chloroplasts light regulation of 32-kd protein synthesis is independent of transcript level.

Spirodela oligorrhiza plants were used to study direct effects of light on plastid gene expression uncoupled from sequential chloroplast‐developmental processes. Specific transcript levels were analysed using chloroplast, gene‐fragment probes. Protein synthesis in vivo and in vitro was also measured. In tissue with mature chloroplasts, light/dark regimes had no substantial effect on transcript levels of the psbA gene coding for the 32‐kd protein. However, under the same conditions, synthesis of the protein itself was dramatically affected. We conclude that in mature chloroplasts synthesis of 32‐kd protein is regulated mainly at a translational level. Transcript levels of the rbcL gene, coding for the large subunit of ribulosebisphosphate carboxylase‐oxygenase, were somewhat sensitive to light/dark effects, although not to the same degree as was synthesis of the protein. We conclude that translational events and transcript levels are involved in regulating synthesis of this polypeptide. Carbon deprivation in the dark reduced psbA and rbcL transcript levels appreciably below those found in non‐starved, dark‐grown tissue. This suggests that starvation and dark effects must be experimentally separated from each other for valid conclusions to be drawn about light/dark regulation of chloroplast gene expression.

Cytoplasmic hybridization in Nicotiana: Mitochondrial DNA analysis in progenies resulting from fusion between protoplasts having different organelle constitutions

SummaryOur previous studies indicated that fusion products with one functional nucleus but organelles of the two fusion partners (i.e. heteroplastomic cybrids) could be obtained by fusing X-irradiated (cytoplasmic donor) with non-irradiated (recipient) Nicotiana protoplasts. The present report deals with the analysis of mitochondria in cybrid populations resulting from the fusion of donor Nicotiana tabacum protoplasts with recipient protoplasts having a N. Sylvestris nucleus but chloroplasts of an alien Nicotiana species, and exhibiting cytoplasmic male sterility. The two fusion parents showed significant differences in restriction patterns of their chloroplast and mitochondrial DNA. Four groups of cybrid plants were obtained by this fusion. All had N. sylvestris nuclei but contained either donor or recipient chloroplasts and had either sterile or fertile anthers. There was no correlation between anther fertility and chloroplasts type. The mitochondrial DNA restriction patterns of sterile cybrids were similar to the respective patterns of the sterile fusion partner while the mitochondrial DNA restriction patterns of the fertile cybrids were similar to the respective patterns of the fertile fusion partner. The results indicate an independent assortment of chloroplasts and mitochondria from the heteroplastomic fusion products.

Molecular Docking Using Surface Complementarity

A method is described to dock a ligand into a binding site in a protein on the basis of the complementarity of the inter‐molecular atomic contacts. Docking is performed by maximization of a complementarity function that is dependent on atomic contact surface area and the chemical properties of the contacting atoms. The generality and simplicity of the complementarity function ensure that a wide range of chemical structures can be handled. The ligand and the protein are treated as rigid bodies, but displacement of a small number of residues lining the ligand binding site can be taken into account. The method can assist in the design of improved ligands by indicating what changes in complementarity may occur as a result of the substitution of an atom in the ligand. The capabilities of the method are demonstrated by application to 14 protein–ligand complexes of known crystal structure.

SPACE: a suite of tools for protein structure prediction and analysis based on complementarity and environment

We describe a suite of SPACE tools for analysis and prediction of structures of biomolecules and their complexes. LPC/CSU software provides a common definition of inter-atomic contacts and complementarity of contacting surfaces to analyze protein structure and complexes. In the current version of LPC/CSU, analyses of water molecules and nucleic acids have been added, together with improved and expanded visualization options using Chime or Java based Jmol. The SPACE suite includes servers and programs for: structural analysis of point mutations (MutaProt); side chain modeling based on surface complementarity (SCCOMP); building a crystal environment and analysis of crystal contacts (CryCo); construction and analysis of protein contact maps (CMA) and molecular docking software (LIGIN). The SPACE suite is accessed at .

Importance of solvent accessibility and contact surfaces in modeling side‐chain conformations in proteins

Contact surface area and chemical properties of atoms are used to concurrently predict conformations of multiple amino acid side chains on a fixed protein backbone. The combination of surface complementarity and solvent‐accessible surface accounts for van der Waals forces and solvation free energy. The scoring function is particularly suitable for modeling partially buried side chains. Both iterative and stochastic searching approaches are used. Our programs (Sccomp‐I and Sccomp‐S), with relatively fast execution times, correctly predict χ1 angles for 92–93% of buried residues and 82–84% for all residues, with an RMSD of ∼1.7 Å for side chain heavy atoms. We find that the differential between the atomic solvation parameters and the contact surface parameters (including those between noncomplementary atoms) is positive; i.e., most protein atoms prefer surface contact with other protein atoms rather than with the solvent. This might correspond to the driving force for maximizing packing of the protein. The influence of the crystal packing, completeness of rotamer library and precise positioning of Cβ atoms on the accuracy of side‐chain prediction are examined. The Sccomp‐S and Sccomp‐I programs can be accessed through the Web (http://sgedg.weizmann.ac.il/sccomp.html) and are available for several platforms.