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Three chiral calcium antagonist drugs, gallopamil and two dihydropyridine derivatives, have been successfully separated within short retention times using both the alpha 1-acid glycoprotein chiral stationary phase (Chiral-AGP) and the ovomucoid column (Ultron ES-OVM). Aqueous buffer at defined pH is modified by the addition of an organic component, in order to modulate the retention properties of each system. Optimization of pH and organic modifier is carried out using the modified simplex method, with Kaiser's peak separation function as a criterion. The influence of pH and percentage of organic modifier on retention, selectivity, resolution and column performance are discussed for the two dihydropyridines analysed on Chiral-AGP and Ultron ES-OVM stationary phases. A new method is proposed as a new chiral system suitability test for these protein-based phases, utilizing a racemic mixture of closely eluting verapamil enantiomers as a probe.

Six chiral stationary phases (CSP) were evaluated for their enantioselectivity towards a series of 45 drugs with different acidic, basic or neutral properties. These CSPs were: a polyacrylamide phase, Chiraspher; two polysaccharide-based phases, cellulose tris-3,5-dimethyl phenylcarbamate (Chiralcel OD) and the S-naphthylethylcarbamate derivative of beta-cyclodextrin (SN-beta-CD; Cyclobond I SN); and three protein-based CSPs--alpha 1-acid glycoprotein (Chiral-AGP), ovomucoid (OVM) and cellulase. A total of 28 different mobile phases were involved. Chiral-AGP, OVM and Chiralcel OD appeared to be the most promising CSPs for the enantio separation of the series of structurally different compounds evaluated. Cellulase and Chiralcel OD show particularly high enantioselectivity towards the group of beta-blocker drugs. The different protein-based CSPs were used in their usual reversed-phase mode. The other phases were used in combination with apolar mobile phases, except for SN-beta-CD, which was evaluated in both modes. Formal optimization strategies were not adopted, although the effect of organic modifier and eluent pH on enantioselectivity was briefly examined for the protein-based phases.

We present a method to search for possible binding modes of molecular fragments at a specific site of a potential drug target of known structure. Our method is based on a Monte Carlo (MC) algorithm applied to the translational and rotational degrees of freedom of the probe fragment. Starting from a randomly generated initial configuration, favorable binding modes are generated using a two-step process. An MC run is first performed in which the energy in the Metropolis algorithm is substituted by a score function that measures the average distance of the probe to the target surface. This has the effect of making buried probes move toward the target surface and also allows enhanced sampling of deep pockets. In a second MC run, a pairwise atom potential function is used, and the temperature parameter is slowly lowered during the run (Simulated Annealing). We repeat this procedure starting from a large number of different randomly generated initial configurations in order to find all energetically favorable docking modes in a specified region around the target. We test this method using two inhibitor-receptor systems: Streptomyces griseus proteinase B in complex with the third domain of the ovomucoid inhibitor from turkey, and dihydrofolate reductase from E. coli in complex with methotrexate. The method could consistently reproduce the complex found in the crystal structure searching from random initial positions in cubes ranging from 25 A to 50 A about the binding site. In the case of SGPB, we were also successful in docking to the native structure. In addition, we were successful in docking small probes in a search that included the entire protein surface.

This review examines the role of protein-bonded chiral stationary phases (CSPs) in enantiomeric separation and investigates the performance characteristics and desired properties of protein CSPs for separation and large-scale operation. The review also discusses the ability of protein-based CSPs to examine the stereochemistry of drug metabolism processes.

The conformations of a polypeptide chain of turkey ovomucoid third domain and its modified form with split reactive site peptide bond Leu-18--Glu-19 have been determined by the literary two-dimensional nuclear Overhauser effect spectroscopy data using an earlier suggested method. It has been found that the polypeptide domain backbone contains an alpha-helical fragment (residues 32-47), five segments having extended conformation (1-5, 11-17, 19-25, 29-31, 48-50) and beta-turn type 1 (26-29). Segments 23-26, 28-31 and 50-51 form an antiparallel beta-structure. Conformational states of the residues entering irregular domain segments have been analysed. Splitting of the reactive site peptide bond Leu-18--Glu-19 is shown to cause insignificant changes in the conformations of a number of amino acid residues except for Val-6 and Asp-7 ones which undergo essential conformational alterations. The conformations of domain in solution and of japanese quail ovomucoid third domain in crystal have been compared. The root-mean-square deviations for phi and psi angles indicate their high similarity. The conformations of turkey ovomucoid third domain and proteinase inhibitor BUSI IIA in solution have been analysed. In spite of moderate (50%) homology of primary structures, some 75% of amino acid residues are shown to have close conformational phi and psi parameters.

We have measured equilibrium constants, Khyd, at pN 6 for the hydrolysis of the reactive site peptide bond (bond between residues 18 and 19) in 42 sequenced variants (39 natural, 3 semisynthetic) of avian ovomucoid third domains. The values range from 0.4 to approximately 35. In 35 cases the effect of a single amino acid replacement on Khyd could be calculated, 13 are without effect and 22 range from a factor of 1.25 to 5.5. Several, but not all, of the effects can be rationalized in terms of residue-residue interactions that are affected by the reactive site hydrolysis. As the measurements are very precise it appears that additional measurements on designed rather than natural variants should allow for the precise measurement of side-chain--side-chain interaction energies.

The effect of internal motion on the quality of a protein structure derived from nuclear magnetic resonance (NMR) cross relaxation has been investigated experimentally. Internal rotation of the tyrosine-31 ring of turkey ovomucoid third domain was found to mediate magnetization transfer; the effect led to underestimation of proton-proton distances in its immediate neighborhood. Experimental methods that distinguish pure cross relaxation from chemical exchange mediated cross relaxation were used to separate true distances from distorted ones. Uncorrected and corrected sets of distances, where the corrections took internal motion into account, each were used as input to a distance geometry program for structural modeling. Each set of distances yielded a family of similar (converged) structures. The two families of structures differed considerably (2 A) in the region of tyrosine-31. In addition, differences as large as 1 A were observed at other positions throughout the structure. These results emphasize the importance of analyzing the effects of internal motions in order to obtain more accurate NMR solution structures.

The urea and heat-induced unfolding-refolding behaviours of chicken egg white ovomucoid and its four fragments representing domains I, II + III, I + II and III were systematically investigated in 0.06 M sodium phosphate buffer (pH 7.0) by difference spectral measurements. The effect of temperature on ovomucoid and its fragments was also studied in 0.05 M sodium acetate buffer (pH 5.0) and in presence of 2 M urea at pH 7.0. Intrinsic viscosity data showed that ovomucoid and its different fragments did not lose any significant amount of their structure under mild acidic conditions (pH 4.6). Difference spectral results showed extensive disruption of the native structure by urea or temperature. Isothermal transitions showed single-step for domain I, domain I + II and domain III, and two-step having one stable intermediate, for ovomucoid and its fragment representing domain II + III. However, the presence of intermediate was not detected when the transitions were studied with temperature at pH 7.0. Strikingly, the single-step thermal transitions of ovomucoid and its fragment representing domain II + III, became two-step when measured either at pH 5.0 or in presence of 2 M urea at pH 7.0. Analysis of the equilibrium data on urea and heat denaturation showed that the second transition observed with ovomucoid or domain II + III represent the unfolding of domain III. The kinetic results of ovomucoid and its fragments indicate that the protein unfolds with three kinetic phases. A comparison of three rate constants for the unfolding of intact ovomucoid with that of its various fragments revealed that domain I, II and III of the protein correspond to the three kinetic phases having rate constants 0.456, 0.120 and 0.054 min-1, respectively. These data have led us to conclude: (i) the unusual stability of ovomucoid towards various denaturants, including temperature, is due to its domain III, (ii) initiation of the folding of the ovomucoid molecule starts from its NH2-terminal region which probably provides the nucleation site for the formation of the subsequent structure and (iii) domains I and II have greater mutual recognition between them as compared to the recognition either of them have with domain III.

Four fragments of ovomucoid representing its individual domains and their different combinations were prepared by peptic and cyanogen bromide cleavages of the protein. The fragments corresponding to domains I + II, II + III, I and III of the parent ovomucoid molecule, were found to be homogeneous by gel filtration and polyacrylamide gel electrophoresis in presence and absence of SDS. Various physico-chemical properties of these proteins, such as molecular weight, NH2- and COOH-terminal amino acid residues, sugar content, isoionic pH, specific extinction coefficient, fluorescence emission spectra, intrinsic viscosity, frictional coefficient, Stokes radius, diffusion coefficient and geometrical mean radius were determined. Analysis of the results on trypsin inhibitory activity of ovomucoid and its different fragments suggested that only domain II is involved in the antitryptic activity of the inhibitor. Optical characteristics of these fragments indicate that they are devoid of tryptophan residues. The hydrodynamic properties suggest that intact ovomucoid and two of its fragments (domain I + II and domain II + III) are significantly different from those of typical globular proteins and are asymmetric in nature. However, the shape of the two remaining fragments representing domains I and III of the intact protein appeared to be compact and globular. Furthermore, domain II of ovomucoid has been suggested to primarily contribute towards the apparent asymmetry in the intact protein.

The three-dimensional structure of a 30-residue synthetic peptide containing the carboxy-terminal "zinc finger" motif of a human enhancer binding protein has been determined by two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. The structure determination is based on 487 approximate interproton distance and 63 torsion angle (phi, psi, and chi 1) restraints. A total of 40 simulated annealing structures were calculated, and the atomic rms distribution about the mean coordinate positions (excluding residues 29 and 30 which are ill-defined) is 0.4 A for the backbone atoms, 0.8 A for all atoms, and 0.41 A for all atoms excluding the lysine and arginine side chains, which are disordered. The solution structure of the zinc finger consists of two irregular antiparallel beta-strands connected by an atypical turn (residues 3-12) and a classical alpha-helix (residues 14-24). The zinc is tetrahedrally coordinated to the sulfur atoms of two cysteines (Cys-5 and Cys-8) and to the N epsilon 2 atoms of two histidines (His-21 and His-27). The two cysteine residues are located in the turn connecting the two beta-strands (residues 5-8); one of the histidine ligands (His-21) is in the alpha-helix, while the second histidine (His-27) is at the end of a looplike structure (formed by the end of the alpha-helix and a turn). The general architecture is qualitatively similar to two previously determined low-resolution Cys2-His2 zinc finger structures, although distinct differences can be observed in the beta-strands and turn and in the region around the two histidines coordinated to zinc. Comparison of the overall polypeptide fold of the enhancer binding protein zinc finger with known structures in the crystallographic data base reveals a striking similarity to one region (residues 23-44) of the X-ray structure of proteinase inhibitor domain III of Japanese quail ovomucoid [Papamokos, E., Weber, E., Bode, W., Huber, R., Empie, M. W., Kato, I., & Laskowski, M. (1982) J. Mol. Biol. 158, 515-537], which could be superimposed with a backbone atomic rms difference of 0.95 A on residues 3-25 (excluding residue 6) of the zinc finger from the enhancer binding protein. The presence of structural homology between two proteins of very different function may indicate that the so-called zinc finger motif is not unique for a class of DNA binding proteins but may represent a general folding motif found in a variety of proteins irrespective of their function.

Plasma desorption mass spectrometry (PDMS), which utilizes the fission fragments from the spontaneous decay of californium-252 to ionize large biomolecules, has emerged as a powerful analytical tool in the field of protein chemistry. Because of its high mass range and sensitivity, PDMS is an excellent method for direct molecular weight determination of peptides and small proteins, with much better accuracy than that of the most commonly used classical methods of gel permeation chromatography or SDS-gel electrophoresis. Furthermore, PDMS analysis of the peptide mixture resulting from a specific enzymatic or chemical cleavage of the protein, allows rapid and facile checking of DNA-derived protein sequences and protein structures produced by recombinant DNA technology. The relatively nondestructive nature of the PD mass spectrometric analysis allows further confirmation of the sequence assignments of individual peptide fragments through additional chemical or enzymatic reactions on the PDMS matrix-bound peptides. This PD mapping approach, combined with classical gas phase sequencing, can be used to identify and locate post-translational modifications in proteins, such as glycosylation, phosphorylation, disulfide linkages, and also detect the presence of peptide and protein variants in synthetic, native and recombinant peptides and proteins.