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We have shown previously that given high-resolution structures of the unbound molecules, structure determination of protein complexes is possible by including biochemical and/or biophysical data as highly ambiguous distance restraints in a docking approach. We applied this method, implemented in the HADDOCK (High Ambiguity Driven DOCKing) package (Dominguez et al., J Am Chem Soc 2003;125:1731-1737), to the targets in the fourth and fifth rounds of CAPRI. Here we describe our results and analyze them in detail. Special attention is given to the role of flexibility in our docking method and the way in which this improves the docking results. We describe extensions to our approach that were developed as a direct result of our participation in CAPRI. In addition to experimental information, we also included interface residue predictions from PPISP (Protein-Protein Interaction Site Predictor; Zhou and Shan, Proteins 2001;44:336-343), a neural network method. Using HADDOCK we were able to generate acceptable structures for 6 of the 8 targets, and to submit at least 1 acceptable structure for 5 of them. Of these 5 submissions, 3 were of medium quality (Targets 10, 11, and 15) and 2 of high quality (Targets 13 and 14). In all cases, predictions were obtained containing at least 40% of the correct epitope at the interface for both ligand and receptor simultaneously.

Protein-protein interaction sites are predicted from a neural network with sequence profiles of neighboring residues and solvent exposure as input. The network was trained on 615 pairs of nonhomologous complex-forming proteins. Tested on a different set of 129 pairs of nonhomologous complex-forming proteins, 70% of the 11,004 predicted interface residues are actually located in the interfaces. These 7732 correctly predicted residues account for 65% of the 11,805 residues making up the 129 interfaces. The main strength of the network predictor lies in the fact that neighbor lists and solvent exposure are relatively insensitive to structural changes accompanying complex formation. As such, it performs equally well with bound or unbound structures of the proteins. For a set of 35 test proteins, when the input was calculated from the bound and unbound structures, the correct fractions of the predicted interface residues were 69 and 70%, respectively.

A homology-based structure prediction method ideally gives both a correct fold assignment and an accurate query-template alignment. In this article we show that the combination of two existing methods, PSI-BLAST and threading, leads to significant enhancement in the success rate of fold recognition. The combined approach, termed COBLATH, also yields much higher alignment accuracy than found in previous studies. It consists of two-way searches both by PSI-BLAST and by threading. In the PSI-BLAST portion, a query is used to search for hits in a library of potential templates and, conversely, each potential template is used to search for hits in a library of queries. In the threading portion, the scoring function is the sum of a sequence profile and a 6x6 substitution matrix between predicted query and known template secondary structure and solvent exposure. "Two-way" in threading means that the query's sequence profile is used to match the sequences of all potential templates and the sequence profiles of all potential templates are used to match the query's sequence. When tested on a set of 533 nonhomologous proteins, COBLATH was able to assign folds for 390 (73%). Among these 390 queries, 265 (68%) had root-mean-square deviations (RMSDs) of less than 8 A between predicted and actual structures. Such high success rate and accuracy make COBLATH an ideal tool for structural genomics.

A survey of 322 proteins showed that the short polar (SP) side chains of four residues, Thr, Ser, Asp, and Asn, have a very strong tendency to form hydrogen bonds with neighboring backbone amides. Specifically, 32% of Thr, 29% of Ser, 26% of Asp, and 19% of Asn engage in such hydrogen bonds. When an SP residue caps the N terminal of a helix, the contribution to helix stability by a hydrogen bond with the amide of the N3 or N2 residue is well established. When an SP residue is in the middle of a helix, the side chain is unlikely to form hydrogen bonds with neighboring backbone amides for steric and geometric reasons. In essence the SP side chain competes with the backbone carbonyl for the same hydrogen-bonding partner (i.e., the backbone amide) and thus SP residues tend to break backbone carbonyl-amide hydrogen bonds. The proposition that this is the origin for the low propensities of SP residues in the middle of alpha helices (relative to those of nonpolar residues) was tested. The combined effects of restricting side-chain rotamer conformations (documented by Creamer and Rose, Proc Acad Sci USA, 1992;89:5937-5941; Proteins, 1994;19:85-97) and excluding side- chain to backbone hydrogen bonds by the helix were quantitatively analyzed. These were found to correlate strongly with four experimentally determined scales of helix-forming propensities. The correlation coefficients ranged from 0.72 to 0.87, which are comparable to those found for nonpolar residues (for which only the loss of side-chain conformational entropy needs to be considered).

A significant fraction of the amino acids in proteins are alpha helical in conformation. Alpha helices in globular proteins are short, with an average length of about twelve residues, so that residues at the ends of helices make up an important fraction of all helical residues. In the middle of a helix, H-bonds connect the NH and CO groups of each residue to partners four residues along the chain. At the ends of a helix, the H-bond potential of the main chain remains unfulfilled, and helix capping interactions involving bonds from polar side chains to the NH or CO of the backbone have been proposed and detected. In a study of synthetic helical peptides, we have found that the sequence Ser-Glu-Asp-Glu stabilizes the alpha helix in a series of helical peptides with consensus sequences. Following the report by Harper and Rose, which identifies SerXaaXaaGlu as a member of a class of common motifs at the N termini of alpha helices in proteins that they refer to as "capping boxes," we have reexamined the side chain-main chain interactions in a variant sequence using 1H NMR, and find that the postulated reciprocal side chain-backbone bonding between the first Ser and last Glu side chains and their peptide NH partners can be resolved. Deletion of two residues N terminal to the Ser-Glu-Asp-Glu sequence in these peptides has no effect on the initiation of helical structure, as defined by two-dimensional (2D) NMR experiments on this variant.(ABSTRACT TRUNCATED AT 250 WORDS)

The multistranded fibers formed by deoxygenated sickle hemoglobin form well ordered spherulitic arrays called domains. Employing laser photolysis of the carboxy derivative to initiate polymerization and domain formation, we have monitored the evolution of the spatial character of domains by light scattering and birefringence. Spatial growth is analyzed by observing a 62.5 microns square area of the sample and storing the image as 2500 discrete elements. Domain formation appears to consist of two phases. In the early phase even symmetric domains are not radially isotropic. Although the domain expands during this period, the internal polymer density as inferred by the light scattering intensity also increases. In the second phase, the domain becomes structurally symmetric and spatially homogeneous, and changes in the scattered intensity including pronounced overshoots occur uniformly across the monitored region. In this phase, the birefringence increases as the scattering decreases, with the same apparent rate.