{beta}-sheet folding of fragment (16-36) of bovine pancreatic trypsin inhibitor as predicted by Monte Carlo simulated annealing

A tertiary structure prediction is described using Monte Carlosimulated annealing for the peptide fragment corresponding toresidues 16–36 of bovine pancreatic trypsin inhibitor(BPTI). The simulation starts with randomly chosen initial conformationsand is performed without imposing experimental constraints usingenergy functions given for generic interatomic interactions.Out of 20 simulation trials, seven conformations show a sheet-likestructure—two strands connected by a turn—althoughthis sheet-like structure is not as rigid as that observed innative BPTI. It is also shown that these conformations are mostlylooped and exhibit a native- like right-handed twist. Unlikethe case with the C-peptide of RNase A, no conspicuous -helicalstructure is found in any of the final conformations obtainedin the simulation. However, the lowest-energy conformation doesnot resemble exactly the native structure. This indicates thatthe rigid ß-sheet conformation of native BPTI merelycorresponds to a local minimum of the energy function if thefragment with residues 16–36 is isolated from the nativeprotein. A statistical analysis of all 20 final conformationssuggests that the tendency for the peptide segments to formextended ß-strands is strong for those with residues18–24, and moderate for those with residues 30–35.The segment of residues 25–29 does not tend to form anydefinite structure. In native BPTI, the former segments areinvolved in the ß-sheet and the latter in the turn.A folding scenario is also speculated from this analysis.     

Evolutionary trace analysis of TGF-{beta} and related growth factors: implications for site-directed mutagenesis

The TGF-ß family of growth factors contains a largenumber of homologous proteins, grouped in several subfamilieson the basis of sequence identity. These subgroups can be combinedinto three broader groups of related cytokines, with markedspecificities for their cellular receptors: the TGF-ßs,the activins and the BMPs/GDFs. Although structural informationis available for some members of the TGF-ß family,very little is known about the way in which these growth factorsinteract with the extra-cellular domains of their multiple cellsurface receptors or with the specific protein inhibitors thoughtto modulate their activity. In this paper, we use the evolutionarytrace method [Lichtarge et al. (1996) J. Mol. Biol., 257, 342–358]to locate two functional patches on the surface of TGF-ß-likegrowth factors. The first of these is centred on a conservedproline (P₃₆ in TGF-ßs 1–3) and contains twoamino acids which could account for the receptor specificityof TGF-ßs (H₃₄ and E₃₅). The second patch is locatedon the other side of the growth factor protomer and surroundsa hydrophobic cavity, large enough to accommodate the side chainof an aromatic residue. In addition to two conserved tryptophansat positions 30 and 32, the main protagonists in this potentialbinding interface are found at positions 31, 92, 93 and 98.Several mutagenesis studies have highlighted the importanceof the C-terminal region of the growth factor molecule in TGF-ßsand of residues in activin A equivalent to positions 31 and94 of the TGF-ßs for the binding of type II receptorsto these ligands. These data, together with our improved knowledgeof possible functional residues, can be used in future structure–functionanalysis experiments.     

A proposed structural model for amyloid fibril elongation: domain swapping forms an interdigitating {beta}-structure polymer

We propose a model illustrating how proteins, which differ intheir overall sequences and structures, can form the propagating,twisted ß-sheet conformations, characteristic of amyloids.Some cases of amyloid formation can be explained through a `domainswapping' event, where the swapped segment is either a ß-hairpinor an unstable conformation which can partially unfold and assumea ß-hairpin structure. As in domain swapping, herethe swapped ß-hairpin is at the edge of the structure,has few (if any) salt bridges and hydrogen bonds connectingit to the remainder of the structure and variable extents ofburied non-polar surface areas. Additionally, in both casesthe swapped piece constitutes a transient `building block' ofthe structure, with a high population time. Whereas in domainswapping the swapped fragment has been shown to be an     

Characterization of the {beta}-lactam binding site of penicillin acylase of Escherichia coli by structural and site-directed mutagenesis studies

The binding of penicillin to penicillin acylase was studiedby X-ray crystallography. The structure of the enzyme–substratecomplex was determined after soaking crystals of an inactiveßN241A penicillin acylase mutant with penicillin G.Binding of the substrate induces a conformational change, inwhich the side chains of     

Synthesis, purification and initial structural characterization of octarellin, a de novo polypeptide modelled on the {alpha}/{beta}-barrel Proteins

We have attempted to construct an artificial polypeptide thatfolds like the eight-stranded parallel ß-barrel structures.Our approach consists of repeating eight times a unit peptidedesigned to adopt a ‘ß-strand/-helix’pattern. A first ‘test’ sequence for this structuralunit was deduced from a series of parameters defined after ananalysis of three natural /ß-barrel proteins and includingprincipally the lengths of the secondary structure elements,the /ß packing and the fitting on average Garnierprofiles. The gene encoding this structural unit was synthesized,cloned and expressed in Escherichia coli either as a monomeror as direct repeats of 2–12 units. Preliminary structuralcharacterization of the 7-, 8- and 9-fold unit polypeptidesby circular dichroism measurements indicates the presence ofthe predicted amount of -helix in the three proteins. Furtheranalysis by urea-gradient gel electrophoresis demonstrates that,in the conditions tested, only the 8-fold unit polypeptide formsa compact structure through a cooperative and rapid two-statefolding transition involving long-range molecular interactions.     

Mix'n'Match: an improved multiple sequence alignment procedure for distantly related proteins using secondary structure predictions, designed to be independent of the choice of gap penalty and scoring matrix

A new multiple sequence alignment procedure is presented. Severaldifferent multiple alignments are made using differing criteria.Having divided the sequences into strongly conserved regions(SCRs) and loosely conserved regions (LCRs), the ‘best’alignment for each LCR is chosen, independently of the otherLCRs, from a selection of possibilities in the multiple alignments.To help make this choice for each LCR, the secondary structureis predicted and shown alongside each different possible alignment.One advantage of this method over automatic, non-interactivemethods, is that the final alignment is not dependent on thechoice of a single set of scoring parameters. Another is that,by allowing interactive choice and by taking account of secondarystructural information, the final alignment is based more onbiological rather than mathematical factors. This method canproduce better alignments than any of the initial automaticmultiple alignment methods used.     

Molecular dynamics model structures for the molten globule state of {alpha}-lactalbumin: aromatic residue clusters I and II

To model the molten globule structure of -lactalbumin, moleculardynamics (MD) simulations were carried out for the protein inexplicit water at high temperature. In these simulations, long-rangeCoulomb interactions were evaluated explicitly with an originalmethod (particle–particle and particle–cell: PPPC)to avoid artifacts caused by the cut-off. The MD simulationswere started from two initial conditions to verify that similarresults would be obtained. From the last 150 ps trajectoriesof the two MD simulations, two partially unfolded average structureswere obtained. These structures had the following common structuralfeatures which are characteristic of the molten globule state.The radii of gyration for these conformations were 7.4 and 9.6%larger than that of the native state. These values were almostthe same as the experimental value (9.6%) observed recentlyby small-angle X-ray scattering (Kataoka,M., Kuwajima,K., Tokunaga,F.and Goto,Y., 1997, Protein Sci., 6, 422–430). Furthermore,aromatic residues of clusters I and II in these structures werefar apart from each other except for Try103–Trp104. Thisresult is in good agreement with NMR experimental results forthe acid-denatured molten globule state (Alexandrescu et al.,1992, 1993); that is, NOE signals between the aromatic residueswere not observed, except for that of Try103–Trp104 inthe molten globule state. Other structural features of thesemodels for the molten globule state are discussed with referenceto native state structures.     

Energetic analysis of binding of progesterone and 5{beta}-androstane-3,17-dione to anti-progesterone antibody DB3 using molecular dynamics and free energy calculations

Molecular dynamics simulations and molecular mechanics–Poisson–Boltzmannsurface area (MM-PBSA) free energy calculations were used tostudy the energetics of the binding of progesterone (PRG) and5ß-androstane-3,17-dione (5AD) to anti-PRG antibodyDB3. Although the two steroids bind to DB3 in different orientations,their binding affinities are of the same magnitude, 1 nM forPRG and 8 nM for 5AD. The calculated relative binding free energyof the steroids, 8.8 kJ/mol, is in fair agreement with the experimentalenergy, 5.4 kJ/mol. In addition, computational alanine scanningwas applied to study the role of selected amino acid residuesof the ligand-binding site on the steroid cross-reactivity.The electrostatic and van der Waals components of the totalbinding free energies were found to favour more the bindingof PRG, whereas solvation energies were more favourable forthe binding of 5AD. The differences in the free energy componentsare due to the binding of the A rings of the steroids to differentbinding pockets: PRG is bound to a pocket in which electrostaticantibody–steroid interactions are dominating, whereas5AD is bound to a pocket in which van der Waals and hydrophobicinteractions dominate.     

Search for the most stable folds of protein chains: II. Computation of stable architectures of {beta}-proteins using a self-consistent molecular field theory

In a preceding paper we presented a novel approach to computationof 3-D folds of protein chains from their amino acid sequences.This approach is a physically correct generalization of the‘threading’ methods. It is based on a self-consistentmolecular field theory and on a physical theory of protein foldingpatterns, which make it possible to examine all the varietyof ‘potentially stable’ folding patterns and allthe variety of the chain conformations within each of them andto determine the thermodynamically stable structure. In thispaper, we apply this approach to single out stable folding patternsand conformations for the chains of ß-sandwich proteinsand show that the similarity of the calculated and observedstructures is usually rather close.