Haemoglobin: the surface buried between the alpha 1 beta 1 and alpha 2 beta 2 dimers in the deoxy and oxy structures

Using the newly available refined co-ordinates of deoxy and oxyhaemoglobin, we have re-examined and compared the interfaces between the dimers alpha 1 beta 1 and alpha 2 beta 2. The most extensive monomer-monomer contacts are between alpha 1 and beta 2, and, symmetrically, alpha 2 and beta 1. In oxyhaemoglobin these interfaces bury 700 A2 less protein surface than in deoxyhaemoglobin. The alpha 1 alpha 2 interface involves similar salt bridges in both forms, but in oxyhaemoglobin buries 240 A2 more surface than in deoxyhaemoglobin. There is a loosely packed beta 1 beta 2 interface burying 320 A2 of surface in oxyhaemoglobin; there is no beta 1 beta 2 interface in deoxyhaemoglobin. The greater stability of the deoxy form, in the absence of ligands, can be attributed to a combination of hydrophobic, van der Waals' and electrostatic interactions.     

Purification of Bacteroides amylophilus protease

A protease was released by Bacteroides amylophilus cells in late stationary phase, approximately 12 hr after maximum cell density was reached. The protease was concentrated by adsorption on diethylaminoethyl (DEAE)-Sephadex and was purified 532-fold by DEAE-Sephadex chromatography, by G-200-Sephadex gel filtration, and by isoelectric focusing. The purified protease was active between pH 4.5 and 12.0 with optima at pH 6.0 and 11.5. Evidence against there being a single protease was given by the differential inhibition of esterase and protease activities by some inhibitors. There was some evidence that only a single protease was present as the ratio of protease activity at various pH values did not alter significantly during purification or when the purified protease was partially heat-inactivated or treated with two specific trypsin-type protease inhibitors: N-alpha-tosyl-l-lysylchloromethyl ketone or phenylmethane-sulfonyl fluoride. Two forms of the same protease were found by acrylamide gel electrophoresis. Gel filtration confirmed the presence of protease in 30,000 and 60,000 molecular-weight forms. Treatment with 1 mm ethylenediaminetetraacetic acid or with 4 m urea failed to convert the 60,000-molecular-weight to the 30,000-molecular-weight species.     

Importance of the release of strand 1C to the polymerization mechanism of inhibitory serpins.

Serpin polymerization is the underlying cause of several diseases, including thromboembolism, emphysema, liver cirrhosis, and angioedema. Understanding the structure of the polymers and the mechanism of polymerization is necessary to support rational design of therapeutic agents. Here we show that polymerization of antithrombin is sensitive to the addition of synthetic peptides that interact with the structure. A 12-m34 peptide (homologous to P14-P3 of antithrombin reactive loop), representing the entire length of s4A, prevented polymerization totally. A 6-mer peptide (homologous to P14-P9 of antithrombin) not only allowed polymerization to occur, but induced it. This effect could be blocked by the addition of a 5-mer peptide with s1C sequence of antithrombin or by an unrelated peptide representing residues 26-31 of cholecystokinin. The s1C or cholecystokinin peptide alone was unable to form a complex with native antithrombin. Moreover, an active antitrypsin double mutant, Pro 361-->Cys, Ser 283-->Cys, was engineered for the purpose of forming a disulfide bond between s1C and s2C to prevent movement of s1C. This mutant was resistant to polymerization if the disulfide bridge was intact, but, under reducing conditions, it regained the potential to polymerize. We have also modeled long-chain serpin polymers with acceptable stereochemistry using two previously proposed loop-A-sheet and loop-C-sheet polymerization mechanisms and have shown both to be sterically feasible, as are "mixed" linear polymers. We therefore conclude that the release of strand 1C must be an element of the mechanism of serpin polymerization.     

Solvent accessibility, protein surfaces, and protein folding.

Studies of the native structures of proteins, together with measurements of the thermodynamic properties of the transition between unfolded and native states, have defined the major components of the forces that stabilize native protein structures. However, the nature of the intermediates in the folding process remains largely hypothetical. It is a fairly widespread and not implausible assumption that the intermediates in the folding of a monomeric protein contain the same kinds of secondary and tertiary structures that appear in the native conformation, and that, although unstable, their lifetimes are prolonged by forces similar to those that stabilize the native structure. We wished to examine what happens if, during the folding of a monomeric protein, regions of secondary structure come together to form an intermediate of reduced instability. We applied calculations of accessible surface area (a measure of hydrophobic stabilization) and parameterized nonbonded energy calculations (measuring the strengths of van der Waals forces) to identify the kinds of stabilizing interactions that might be available to such an intermediate. First, we analyzed the total buried surface area of two types of proteins into contributions from formation of secondary structure alone, interaction of pairs of secondary-structural elements, the formation of the structure alone, interaction of pairs of secondary-structural elements, the formation of the complete secondary structure without the turns, and the complete native structure. The formation of secondary structure alone, without tertiary-structural interactions, buries roughly half the surface that the complete structure does. We then analyzed in more detail the approach of two alpha-helices to form a complex, as an illustrative example of the nature of the interaction between compact structural units which remain fairly rigid during their interaction. Many features of the results are not limited to the interaction of alpha-helices. (The results therefore neither confirm nor refute the hypothesis that alpha-helices are intermediates in the folding proteins). We find that the first forces to be felt upon approach arise from solvent conditions on the relative position and orientation of the two helices as does the close packing which optimizes the van der Waals interactions at shorter distances apart. Therefore there appears to be a range of distances in which hydrophobic interactions could create a nonspecific complex between two helices in which the side chains might have sufficient time to seek the proper interdigitation observed in the native structure, where the two helices are in intimate contact. Indeed, we find that only in the final stages of approach is the native geometry the most stable; in the region in which solvent-exclusion forces predominate, the conformation with helix axes parallel is more stable than the native conformation, in the cases we examined...     

Structural repertoire of the human VH segments.

The VH gene segments produce the part of the VH domains of antibodies that contains the first two hypervariable regions. The sequences of 83 human VH segments with open reading frames, from several individuals, are currently known. It has been shown that these sequences are likely to form a high proportion of the total human repertoire and that an individual's gene repertoire produces about 50 VH segments with different protein sequences. In this paper we present a structural analysis of the amino acid sequences produced by the 83 segments. Particular residue patterns in the sequences of V domains imply particular main-chain conformations, canonical structures, for the hypervariable regions. We show that, in almost all cases, the residue patterns in the VH segments imply that the first hypervariable regions have one of three different canonical structures and that the second hypervariable regions have one of five different canonical structures. The different observed combinations of the canonical structures in the first and second regions means that almost all sequences have one of seven main-chain folds. We describe, in outline, structures of the antigen binding site loops produced by nearly all the VH segments. The exact specificity of the loops is produced by (1) sequence differences in their surface residues, particularly at sites near the centre of the combining site, and (2) sequence differences in the hypervariable and framework regions that modulate the relative positions of the loops.     

Probing protein structure by solvent perturbation of nuclear magnetic resonance spectra. Nuclear magnetic resonance spectral editing and topological mapping in proteins by paramagnetic relaxation filtering.

Soluble spin labels, which "bleach" the surface proton resonances of a protein to n.m.r. measurements, can provide useful information about protein conformation and dynamics. The use of the soluble nitroxide, TEMPOL, has been explored to show the correlation of the paramagnetic perturbations of protein two-dimensional n.m.r. data with proton exposure to the free radical in hen egg-white lysozyme. The results demonstrate that the nitroxide approaches the protein randomly, and that the extent of the observed paramagnetic effects reflects the native folding pattern of the protein. A correlation of spectral simplification with the known tertiary structure establishes the feasibility of new strategies for topological mapping of surface and buried protons of the protein. Application to the elucidation of protein structure and to the study of dynamical processes is discussed.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Biodiversity of 52 chicken populations assessed by microsatellite typing of DNA pools

In a project on the biodiversity of chickens funded by the European Commission (EC), eight laboratories collaborated to assess the genetic variation within and between 52 populations from a wide range of chicken types. Twenty-two di-nucleotide microsatellite markers were used to genotype DNA pools of 50 birds from each population. The polymorphism measures for the average, the least polymorphic population (inbred C line) and the most polymorphic population (Gallus gallus spadiceus) were, respectively, as follows: number of alleles per locus, per population: 3.5, 1.3 and 5.2; average gene diversity across markers: 0.47, 0.05 and 0.64; and proportion of polymorphic markers: 0.91, 0.25 and 1.0. These were in good agreement with the breeding history of the populations. For instance, unselected populations were found to be more polymorphic than selected breeds such as layers. Thus DNA pools are effective in the preliminary assessment of genetic variation of populations and markers. Mean genetic distance indicates the extent to which a given population shares its genetic diversity with that of the whole tested gene pool and is a useful criterion for conservation of diversity. The distribution of population-specific (private) alleles and the amount of genetic variation shared among populations supports the hypothesis that the red jungle fowl is the main progenitor of the domesticated chicken.     

Modularity and homology: modelling of the titin type I modules and their interfaces

Titin is a giant muscle protein with a highly modular architecture consisting of multiple repeats of two sequence motifs, named type I and type II. Type I motifs are homologous to members of the fibronectin type 3 (Fn3) superfamily, one of the motifs most widespread in modular proteins. Fn3 domains are thought to mediate protein-protein interactions and to act as spacers. In titin, Fn3 modules are present in two different super-repeated patterns, likely to be involved in sarcomere assembly through interactions with A-band proteins. Here, we discuss results from homology modelling the whole family of Fn3 domains in titin. Homology modelling is a powerful tool that will play an increasingly important role in the post-genomic era. It is particularly useful for extending experimental structure determinations of parts of multidomain proteins that contain multiple copies of the same motif. The 3D structures of a representative titin type I domain and of other extracellular Fn3 modules were used as a template to model the structures of the 132 copies in titin. The resulting models suggest residues that contribute to the fold stability and allow us to distinguish these from residues likely to have functional importance. In particular, analysis of the models and mapping of the consensus sequence onto the 3D structure suggest putative surfaces of interaction with other proteins. From the structures of isolated modules and the pattern of conservation in the multiple alignment of the whole titin Ig and Fn3 families, it is possible to address the question of how tandem modules are assembled. Our predictions can be validated experimentally.