Combined-information protein structure refinement: Potential energy-constrained real-space method for refinement with limited diffraction data.

A potential energy-constrained real-space refinement method designed for use with x-ray diffraction data of low to moderate resolution has been developed. The number of adjustable parameters is severely restricted to ensure a reasonable ratio of data to parameters. Only dihedral angles are allowed to vary; bond lengths and bond angles are fixed at physically reasonable values.

Conformations of cyclo(L-alanyl-L-alanyl-epsilon-aminocaproyl) and of cyclo(L-alanyl-D-alanyl-epsilon-aminocaproyl); cyclized dipeptide models for specific types of beta-bends.

Conformational energy calculations indicate that the peptide backbones of the low-energy conformations of the cyclized dipeptide derivatives cyclo (L-alanyl-L-alanyl-epsilon-aminocaproyl) and cyclo (L-alanyl-D-alanyl-epsilon-aminocaproyl) are constrained to form beta-bends of types I + III and II, respectively. Thus, the two compounds can serve as models for the spectroscopic properties of beta-bends of these types. The coupling constants obtained from 1H n.

Conformational energy analysis of melanostatin.

Conformational energy calculations were carried out on the hypothalamic hormone melanostatin, a tripeptide with the primary structure H-L-Pro-L-Leu-Gly-NH2. The calculated lowest energy conformation was a type II beta bend, very similar to that reported in an X-ray crystal study. This conformation, however, was only one of 109 low-energy structures (less than or equal to 3 kcal/mol above the global minimum), indicating that the molecule in solution exists as an ensemble of conformations and is very flexible, in agreement with relaxation data from n.

Introduction of short-range restrictions in a protein-folding algorithm involving a long-range geometrical restriction and short-, medium-, and long-range interactions.

A protein-folding algorithm, based on short-range and geometrical long-range restrictions, is applied to bovine pancreatic trypsin inhibitor (BPTI). These restrictions are used to define a starting conformation, SI, by means of a space-filling model of the protein, whose energy is then minimized. The long-range restriction is the imposition of the native spatial geometric arrangement of the loops (SGAL) formed by the disulfide bonds.

Regeneration of ribonuclease A from the reduced protein. Isolation and identification of intermediates, and equilibrium treatment.

Reduced RNase A was reoxidized, and the incorrectly formed disulfide bonds were reshuffled to the native ones by oxidized and reduced glutathiones, as described in the first paper of this series. The intermediates in the regeneration of the disulfide bonds were trapped without any chemical modification and were fractionated on a carboxymethylcellulose column at pH 3.5 with a salt gradient.

Conformational characteristics of the N-acetyl-N'-methylamides of the four (Lys, Tyr) dipeptides.

The conformational properties of the N-acetyl-N'-methylamides of the dipeptides lysyl-lysine, lysyl-tyrosine, tyrosyl-lysine, and tyrosyl-tyrosine were studied by means of conformational energy calculations, by n.m.r.

Resonance Raman spectroscopy of arsanilazocarboxypeptidase A: mode of inhibitor binding and active-site topography.

The interaction of inhibitors with the active site of arsanilazocarboxypeptidase A has been investigated by means of resonance Raman spectroscopy. The resonance Raman bands of the active-site azotyrosine-248 residue have been shown previously to be sensitive to its state of ionization and its interactions with nearby groups. In particular, the azophenol form of azotyrosine-248 can adopt two different coexisting conformations that differ with respect to the presence or absence of an intramolecular hydrogen bond between the phenolic proton and a nitrogen atom of the azo group.

Spectroscopic study of the conformations of proline-containing oligopeptides in the crystalline state and in solution.

A conformational study has been carried out on a series of linear proline-containing oligopeptides (ZGP, ZGPL, ZGPLG and ZGPLGP) in both the crystalline state and in DMSO-d6 solution, using Raman and n.m.r.

Intermolecular anti-parallel beta sheet: Comparison of predicted and observed conformations of gramicidin S.

A recently determined x-ray structure of the hydrated gramicidin S-urea complex is compared with a structure predicted by conformational energy minimization. It is shown that the two structures are in good general agreement, including the prediction of a hydrogen bond between the side-chain amino group of ornithine and the backbone carbonyl of phenylalanine. This agreement demonstrates the power of empirical potential energy methods in conformational analysis and illustrates one method for solution of the multiple-minimum problem for small peptides.

Study of the state of ionization of [Leu5]-enkephalin in the crystal and in solution.

The state of ionization of [Leu5]-enkephalin in the crystal and in solution was studied with the aid of Raman, i.r., and n.

Conformational study of [Leu5]-enkephalin by laser Raman spectroscopy.

The Raman spectrum of [Leu5]-enkephalin in the solid state indicates the presence of a type I' beta bend, in agreement with X-ray data of Smith & Griffin (1978). In solution, this molecule takes on different conformations in different solvents. In DMSO-d6, it forms a beta-bend structure whereas in water it exists as an ensemble of conformations.

Mechanism of action of thrombin on fibrinogen. Size of the A alpha fibrinogen-like peptide that contacts the active site of thrombin.

The following peptides were synthesized by classical methods in solution: Ac-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-NHCH3 (F-4), Ac-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-NHCH3 (F-5), and Ac-Phe-Leu-Ala-Glu-Glv-Gly-Gly-Gly-Val-Arg-Gly-Pro-NHCH3 (F-6). The rates of hydrolysis of the Arg-Gly bond in these peptides by thrombin were measured, and the values of the specificity constant, kcat/KM, were all found to be approximately 2 X 10(-7) [(NIH unit/L)s]-1, similar to that for a peptide (F-3) having an additional Arg residue between Glu- and -NHCH3 of F-4. The difference between this value and that for the A alpha chain of bovine fibrinogen is attributed to slight conformational differences arising from long-range interactions present in fibrinogen but not in the synthetic peptides.

Preferred conformation of the tert-butoxycarbonyl-amino group in peptides.

Structural parameters, derived from X-ray crystallographic data, have been compiled for amino acid and linear peptide derivatives which contain the N-terminal tert-butoxycarbonyl (Boc) group or its next higher homolog, the tert-amyloxycarbonyl group. The comparison of the geometry of the urethane group in Boc-derivatives with that of the peptide group shows small differences in bond angles about the trigonal carbon, because of altered interactions when a C alpha H group of a peptide unti is replaced by an ester oxygen. In contrast to the strong preference of the peptide bond for the trans form (except when it precedes proline), the urethane amide bond adopts both the cis and trans conformations in crystals.

Location of peptide fragments in the fibrinogen molecule by immunoelectron microscopy.

Antibodies to the disulfide knot fragment of bovine fibrinogen have been used to locate the site of this fragment within the intact fibrinogen molecule. The antibodies were isolated from rabbit antifibrinogen antisera by affinity chromatography. Electron micrographs of reaction mixtures of bovine fibrinogen and antibodies against the disulfide knot fragment showed pairs of fibrinogen molecules crosslinked by antibody molecules as well as higher order antibody-fibrinogen complexes.

Conformational studies of somatostatin and selected analogues by Raman spectroscopy.

Laser Raman spectroscopy has been used to study the conformations of somatostatin and some selected analogues in aqueous solution. The results indicate that the CS-SC dihedral angles of somatostatin and of these analogues (except [Ala3,14]-SS, which has no disulfide bond) are within 20 degrees of +/- 85 degrees, and the SS-CC dihedral angles are predominantly in the range of 50 degrees-180 degrees. Furthermore, from the behavior of the amide I' and amide III bands, it appears that somatostatin adopts a beta-pleated sheet structure, whereas its analogues are less ordered (to varying degrees).