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I D Kuntz

Publications and source records attributed to I D Kuntz.

At least 19 recordsLinked to original sources
PubMed1992

Sequence-specific 1H and 15N resonance assignments for both equilibrium forms of the soluble heme binding domain of rat ferrocytochrome b5.

15N and 1H resonance assignments for backbone and side-chain resonances of both equilibrium forms of rat ferrocytochrome b5 have been obtained, using 15N-1H heteronuclear correlation methods employing globally 15N-labeled protein. Unlike other cytochrome b5 species assigned to date (Guiles et al., 1990) the rat cytochrome exists as an equilibrium distribution of conformers in nearly equal abundance (Lee et al., 1990). The ratio of conformers present in all other species variants is approximately 1:9. More than 40% of all residues of the rat protein exhibit NMR-detectable heterogeneity due to the 180 degrees rotation of the heme about the alpha, gamma-meso axis. NOESY and HOHAHA relayed 15N-1H double-DEPT heteronuclear correlation methods were an indispensible tool for the deconvolution of a system with this level of heterogeneity. Differences in the resonance assignments between the two equilibrium conformers were found to be as great as differences between species variants we have previously reported. On the basis of the magnitude and extent of the observed chemical shift differences and specific NOESY connectivities observed in the two isomers, we believe the two equilibrium conformers differ not only by a simple back-to-front flip of the heme but also by an additional rotation about an axis normal to the heme plane as has been previously suggested by Pochapsky et al. (1990). A short segment of the protein at the N-terminus could not be assigned, presumably due to rapid exchange of solvent-accessible amide protons in this disordered segment of the protein. Assignments for 93 of the 98 residues of this 12-kDa protein have been obtained.

Animals↗
PubMed1992

Structure-based strategies for drug design and discovery.

Most drugs have been discovered in random screens or by exploiting information about macromolecular receptors. One source of this information is in the structures of critical proteins and nucleic acids. The structure-based approach to design couples this information with specialized computer programs to propose novel enzyme inhibitors and other therapeutic agents. Iterated design cycles have produced compounds now in clinical trials. The combination of molecular structure determination and computation is emerging as an important tool for drug development. These ideas will be applied to acquired immunodeficiency syndrome (AIDS) and bacterial drug resistance.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Disulfide bond-coupled folding of bovine pancreatic trypsin inhibitor derivatives missing one or two disulfide bonds.

The disulfide bond-coupled folding and unfolding mechanism (at pH 8.7, 25 degrees C in the presence of oxidized and reduced dithiothreitol) was determined for a bovine pancreatic trypsin inhibitor mutant in which cysteines 30 and 51 were replaced with alanines so that only two disulfides, between cysteines 14 and 38 and cysteines 5 and 55, remain. Similar studies were made on a chemically-modified derivative of the mutant retaining only the 5-55 disulfide. The preferred unfolding mechanism for the Ala30/Ala51 mutant begins with reduction of the 14-38 disulfide. An intramolecular rearrangement via thiol-disulfide exchange, involving the 5-55 disulfide and cysteines 14 and/or 38, then occurs. At least five of six possible one-disulfide bond species accumulate during unfolding. Finally, the disulfide of one or more of the one-disulfide bond intermediates (excluding that with the 5-55 disulfide) is reduced giving unfolded protein. The folding mechanism seems to be the reverse of the unfolding mechanism; the observed folding and unfolding reactions are consistent with a single kinetic scheme. The rate constant for the rate-limiting intramolecular folding step--rearrangements of other one-disulfide bond species to the 5-55 disulfide intermediate--seems to depend primarily on the number of amino acids separating cysteines 5 and 55 in the unfolded chain. The energetics and kinetics of the mutant's folding mechanism are compared to those of wild-type protein [Creighton, T. E., & Goldenberg, D. P. (1984) J. Mol. Biol. 179, 497] and a mutant missing the 14-38 disulfide [Goldenberg, D. P. (1988) Biochemistry 27, 2481]. The most striking effects are destabilization of the native structure and a large increase in the rate of unfolding.

Alanine↗
PubMed1992

Comparison of solution structures of mutant bovine pancreatic trypsin inhibitor proteins using two-dimensional nuclear magnetic resonance.

Structural perturbations due to a series of mutations at the 30-51 disulfide bond of bovine pancreatic trypsin inhibitor have been explored using NMR. The mutants replaced cysteines at positions 30 and 51 by alanine at position 51 and alanine, threonine, or valine at position 30. Chemical shift changes occur in residues proximate to the site of mutation. NOE assignments were made using an automated procedure, NASIGN, which used information from the wild-type crystal structure. Intensity information was utilized by a distance geometry algorithm, VEMBED, to generate a series of structures for each protein. Statistical analyses of these structures indicated larger averaged structural perturbations than would be expected from crystallographic and other information. Constrained molecular dynamics refinement using AMBER at 900 K was useful in eliminating structural movements that were not a necessary consequence of the NMR data. In most cases, statistically significant movements are shown to be those greater than approximately 1 A. Such movements do not appear to occur between wild type and A30A51, a result confirmed by crystallography (Eigenbrot, C., Randal, M., & Kossiakoff, A.A., 1990, Protein Eng. 3, 591-598). Structural alterations in the T30A51 or V30A51 mutant proteins near the limits of detection occur in the beta-loop (residues 25-28) or C-terminal alpha-helix, respectively.

Amino Acid Sequence↗
PubMed1992

A fast and efficient method for 2D and 3D molecular shape description.

A new formalism for molecular shape description is described. The formalism, based on considering each molecule as a collection of its 3-atom submolecules, is applied to both the graph theory and geometrical coordinate representations of molecules. The timing results for shape description of several databases indicate that this new method is applicable to large databases. Furthermore, results from clustering a small database show good agreement with clustering results obtained by a distance-matching algorithm.

Algorithms↗
PubMed1992

Automated site-directed drug design using molecular lattices.

Receptor-based drug design is predicated on the knowledge of the structure of a target receptor and the principles of molecule recognition. The objective is to produce a wide diversity of structures that are sterically and electrostatically complementary to a specified receptor site. Many drug-receptor interactions are controlled by a few key receptor groups. This observation leads to a design approach in which one focuses on chemical fragments that putatively interact with the key receptor groups. There then remains the difficult task of joining the fragments into molecular structures that match the spatial patterns of recognition forces in the receptor site. In this paper, we describe a new modeling program, BUILDER, that combines database searching techniques and structure generation algorithms within an interactive graphics modeling environment (MidasPlus). A novel tool for process communication (delegate) is introduced and examples of its use are given. To demonstrate the functionality of the package and its ability to produce novel structures, we examine the active site of HIV-1 protease.

Algorithms↗
PubMed1991

Protein docking and complementarity.

Predicting the structures of protein-protein complexes is a difficult problem owing to the topographical and thermodynamic complexity of these structures. Past efforts in this area have focussed on fitting the interacting proteins together using rigid body searches, usually with the conformations of the proteins as they occur in crystal structure complexes. Here we present work which uses a rigid body docking method to generate the structures of three known protein complexes, using both the bound and unbound conformations of the interacting molecules. In all cases we can regenerate the geometry of the crystal complexes to high accuracy. We also are able to find geometries that do not resemble the crystal structure but nevertheless are surprisingly reasonable both mechanistically and by some simple physical criteria. In contrast to previous work in this area, we find that simple methods for evaluating the complementarity at the protein-protein interface cannot distinguish between the configurations that resemble the crystal structure complex and those that do not. Methods that could not distinguish between such similar and dissimilar configurations include surface area burial, solvation free energy, packing and mechanism-based filtering. Evaluations of the total interaction energy and the electrostatic interaction energy of the complexes were somewhat better. Of the techniques that we tried, energy minimization distinguished most clearly between the "true" and "false" positives, though even here the energy differences were surprisingly small. We found the lowest total interaction energy from amongst all of the putative complexes generated by docking was always within 5 A root-mean-square of the crystallographic structure. There were, however, several putative complexes that were very dissimilar to the crystallographic structure but had energies that were close to that of the low energy structure. The magnitude of the error in energy calculations has not been established in macromolecular systems, and thus the reliability of the small differences in energy remains to be determined. The ability of this docking method to regenerate the crystallographic configurations of the interacting proteins using their unbound conformations suggests that it will be a useful tool in predicting the structures of unsolved complexes.

Algorithms↗
PubMed1991

Molecular dynamics simulations of small peptides: dependence on dielectric model and pH.

There has been much interest recently in the structure of small peptides in solution. A recent study by Bradley and co-workers [(1989) in Techniques of Protein Chemistry, Hugli, T.E., Ed., Academic Press, Orlando, FL, pp. 531-546; (1990) Journal of Molecular Biology, 215, pp. 607-622] describes a 17-residue peptide that is stable as a monomeric helix in aqueous solution at low pH, as determined by two-dimensional nmr and CD spectroscopy. They also have determined the helix content of the peptide as a function of pH using CD. We performed molecular dynamics simulations, with an empirical force field, of this peptide at low pH, with three different dielectric models: a linear distance-dependent dielectric function (epsilon = R); a modified form [J. Ramstein and R. Lavery (1988) Proceedings of the National Academy of Science, USA, Vol. 85, pp. 7231-7235] of the sigmoidal distance-dependent dielectric function of Hingerty and co-workers [(1985) Biopolymers, Vol. 24, pp. 427-439]; and epsilon = 1 with the peptide immersed in a bath of water molecules. We found that simulations with the sigmoidal dielectric function and the model with explicit water molecules resulted in average distances for particular interactions that were consistent with the experimental nmr results, with the sigmoidal function best representing the data. However, these models exhibited very different helix-stabilizing interactions. We also performed simulations using the sigmoidal function at moderate and high pH to compare to experimental determinations of the pH dependence of helix content. Helix content did not decrease with increases in pH, as shown experimentally. We did, however, observe changes in a specific side chain-helix dipole interaction that was implicated in determining the pH-dependent behavior of this peptide. Overall, the sigmoidal dielectric function was a reasonable alternative to adding explicit water molecules. In comparing 100 ps molecular dynamics simulations, the sigmoidal function was much less computer intensive and sampled more of conformational space than the treatment using explicit water molecules. Sampling is especially important for this system since the peptide has been shown experimentally to populate both helical and nonhelical conformations.

Amino Acid Sequence↗
PubMed1991

Secretion incompetence of bovine pancreatic trypsin inhibitor expressed in Escherichia coli.

There is increasing evidence that protein folding and protein export are competing processes in prokaryotic cells. Virtually all secretion studies reported to date, however, have employed proteins that are relatively uncharacterized in terms of their folding behavior and three-dimensional structure. In contrast, the structural and biochemical parameters governing the folding of bovine pancreatic trypsin inhibitor (BPTI) and several of its mutants have been studied intensively. We therefore undertook a study of the secretion behavior in Escherichia coli of recombinant BPTI and its mutants. Wild-type BPTI and two well-characterized folding mutants (C14A, C38A)BPTI and (C30A, C51A)BPTI (missing the 14-38 and 30-51 disulfide bonds, respectively), were investigated by analyzing their expression fused to an E. coli signal sequence or to two synthetic IgG-binding domains of staphylococcal protein A. Both disulfide mutants are destabilized relative to wild-type BPTI and exhibit markedly altered folding kinetics: one (C14A, C38A) folds more slowly than wild-type BPTI and the other (C30A, C51A) unfolds more rapidly. Both mutants were observed to be exported 3-10 times more efficiently than the wild-type molecule. Moreover, the levels of unprocessed preprotein in the cytoplasm were severalfold higher for the wild-type fusion than for the fusion to the two folding mutants. Intracellular degradation of the BPTI moiety was also observed. These results are consistent with traffic of intracellular BPTI preproteins on at least three routes along the secretory pathway: (a) facile secretion of unfolded material, (b) intracellular folding leading to secretion blockage, and (c) degradation followed by export of truncated molecules. A novel feature of these findings is the implication that disulfide bonds can form in the bacterial cytoplasm and lead to secretion incompetence.

Amino Acid Sequence↗
PubMed1991

Effects of limited input distance constraints upon the distance geometry algorithm.

In this paper we examine the distance geometry (DG) algorithm in the form used to determine the structure of proteins. We focus on three aspects of the algorithm: bound smoothing with the triangle inequality, the random selection of distances within the bounds, and the number of distances needed to specify a structure. Computational experiments are performed using simulated and real data for basic pancreatic trypsin inhibitor (BPTI) from nmr and crystallographic measurements. We find that the upper bounds determined by bound smoothing to be a linear function of the true crystal distance. A simple model that describes the results obtained with randomly selected trial distances is proposed. Using this representation of the trial distances, we show that BPTI DG structures are more compact than the true crystal structure. We also show that the DG-generated structures no longer resemble test structures when the number of these interresidue distance constraints is less than the number of degrees of freedom of the protein backbone. While the actual model will be sensitive the way distances are chosen, our conclusions are likely to apply to other versions of the DG algorithm.

Algorithms↗
PubMed1991

A molecular dynamics simulation of polyalanine: an analysis of equilibrium motions and helix-coil transitions.

An understanding of helix dynamics can aid in interpreting the motions of proteins. The conformational transitions that occur also appear to play a role in protein folding. Structural studies of isolated peptides in solution are just becoming available. However, detailed analysis of the helix-coil transition is still not available and will be difficult to obtain experimentally. For these reasons, we performed a long molecular dynamics simulation of polyalanine at high temperature. Using this approach, we obtain a description of the overall structure and inherent flexibility of the chain as well as a structural picture of the conformational changes that occur. In this way, we can address both equilibrium properties of the peptide and the dynamics and mechanisms of the structural transitions. Our results correlate fairly well with the available experimental data and previous simulations aimed at addressing alpha-helix dynamics. The peptide spends the bulk of its time fluctuating between different conformations with intermediate helix contents. Transitions between highly ordered and highly disordered structures were rare, but they occurred rapidly. Our distribution of conformations favored collapsed states. Hence, our transitions to structures with high helical content were from fluctuating compact structures. The conversion between helix and coil occurred sequentially on a residue-by-residue basis. However, there was local cooperativity; the transition of a residue to the coil state was facilitated after a neighboring group became nonhelical. The relevance of our results to protein folding is also discussed.

Models, Chemical↗
PubMed1991

Confirmation of the predicted source of a slow folding reaction: proline 8 of bovine pancreatic trypsin inhibitor.

A major question in protein structural analysis concerns the applicability of results from model systems to other proteins. Theoretical approaches seem the best manner of transferring information from one system to another, but their accuracy in the model systems must first be tested with results from experiment. Since bovine pancreatic trypsin inhibitor (BPTI) is a model system for the evaluation of energy minimization and molecular dynamics routines, we can use folding and stability measurements to examine the reliability of these methods. All two-disulfide mutants of BPTI investigated thus far have two very slow folding reactions which have characteristics of proline isomerization. These reactions may occur because the non-native cis form of two of the four prolines in BPTI significantly destabilizes the folded state of the protein. Previous energy minimization studies of wild-type BPTI suggested that the cis form of Pro8 was the most destabilizing of the four prolines [Levitt,M. (1981) J. Mol. Biol., 145, 251-263]. In this paper, we show that mutation of Pro8----Gln in the two-disulfide bond mutant Val30/Ala51 results in a loss of the slowest folding reaction, consistent with Levitt's prediction.

Animals↗
PubMed1991

Intracellular expression of BPTI fusion proteins and single column cleavage/affinity purification by chymotrypsin.

The novel and efficient expression system described here produces formerly poorly expressed, proteolytically unstable mutants of bovine pancreatic trypsin inhibitor (BPTI). A new, single column method for the cleavage of a recombinant fusion to BPTI and affinity purification of the BPTI moiety by immobilized chymotrypsin is an integral part of the system. Wild-type and mutant BPTI molecules are expressed in Escherichia coli as fusion proteins forming intracellular inclusion bodies. Transcription initiation is under the control of the E. coli trp promoter. The expressed protein is tripartite fusion comprising (i) a portion of the TrpLE leader peptide, (ii) a synthetic IgG binding domain derived from protein A and (iii) the BPTI variant. Solubilization of the inclusion bodies and refolding of the fusion proteins in a thiol-disulfide shuffling system yields correctly folded inhibitor molecules. In the single column purification and cleavage procedure, immobilized chymotrypsin cleaves the refolded fusion protein and releases affinity purified active BPTI mutants with correct N-termini. Mutant BPTI molecules which do not fold into active inhibitors are also stably expressed in inclusion bodies but cannot be purified by this method. Unlike previously described secretion systems for the production of BPTI, expression levels in this system appear to be independent of both the mutation in the BPTI gene and the activity of the expressed protein. Mutants poorly expressed in secretion systems can now be produced in sufficient quantities for protein folding studies and structural analysis using X-ray crystallography and NMR spectroscopy.

Amino Acid Sequence↗
PubMed1990

Studies of synthetic helical peptides using circular dichroism and nuclear magnetic resonance.

We have designed a set of 17-residue synthetic peptides to be monomeric helices in aqueous solution. Circular dichrosim experiments indicate the presence of helical structure in aqueous solution at low temperature and low pH. The two-dimensional nuclear magnetic resonance results for one of the peptides show a segment of ten residues which clearly meets all of the criteria for the existence of helical structure at both 5 degrees C and 15 degrees C. The first four residues of the peptide are in a largely extended conformation. Calculations suggest that residues 5 through 14 are significantly helical at 5 degrees C. When the temperature is increased, circular dichroism spectra indicate that the helical content decreases. At 15 degrees C, the 3JN alpha coupling constants increase in the helical region, indicating an increase in motion or conformational averaging in the helical segment. None of the peptides has pH titration behavior consistent with salt bridge stabilization of helical conformation. Our data lend themselves to interpretation with the helix dipole model and specific side-chain interactions. When the N and C termini charges are removed the helical content of the peptides increases. The amount of helicity increases as the pH is lowered, due to the ionization of His16. Much of the helical stabilization appears to be due to a specific side-chain interaction between His16 and Tyr12.

Amino Acid Sequence↗
PubMed1990

Denaturant-dependent folding of bovine pancreatic trypsin inhibitor mutants with two intact disulfide bonds.

The equilibrium and kinetic behavior of the guanidine hydrochloride (Gdn-HCl) induced unfolding/refolding of four bovine pancreatic trypsin inhibitor (BPTI) mutants was examined by using ultraviolet difference spectroscopy. In three of the mutants, we replaced the buried 30-51 disulfide bond with alanine at position 51 and valine (Val30/Ala51), alanine (Ala30/Ala51), or threonine (Thr30/Ala51) at position 30. For the fourth mutant, the solvent-exposed 14-38 disulfide was substituted by a pair of alanines (Ala14/Ala38). All mutants retained the 5-55 disulfide. Experiments were performed under oxidizing conditions; thus, both the unfolded and folded forms retained two native disulfide bonds. Equilibrium experiments demonstrated that all four mutants were destabilized relative to wild-type BPTI. However, the stability of the 30-51 mutants increased with the hydrophobicity of the residue substituted at position 30. Kinetic experiments showed that all four mutants contained two minor slow refolding phases with characteristics of proline isomerization. The specific behavior of the phases depended on the location of the disulfide bonds. The major unfolding/refolding phase for each of the 30-51 mutants was more than an order of magnitude slower than for Ala14/Ala38 or for BPTI in which the 14-38 disulfide bond was specifically reduced and blocked with iodoacetamide [Jullien, M., & Baldwin, R. L. (1981) J. Mol. Biol. 145, 265-280]. Since this effect is independent of the stability of the protein, it is consistent with a model in which the proper docking of the interior residues of the protein is the rate-limiting step in the folding of these mutants.

Amino Acid Sequence↗
PubMed1990

Structural studies of cytochrome b5: complete sequence-specific resonance assignments for the trypsin-solubilized microsomal ferrocytochrome b5 obtained from pig and calf.

We report complete sequence-specific proton resonance assignments for the trypsin-solubilized microsomal ferrocytochrome b5 obtained from calf liver. In addition, sequence-specific resonance assignments for the main-chain amino acid protons (i.e., C alpha, C beta, and amide protons) are also reported for the porcine cytochrome b5. Assignment of the majority of the main-chain resonances was rapidly accomplished by automated procedures that used COSY and HOHAHA peak coordinates as input. Long side chain amino acid spin system identification was facilitated by long-range coherence-transfer experiments (HOHAHA). Problems with resonance overlap were resolved by examining differences between the two-dimensional 500-MHz NMR spectra of rabbit, pig, and calf proteins and by examining the temperature-dependent variation of amide proton resonances. Calculations of the aromatic ring-current shifts for protons that the X-ray crystal structure indicated were proximal to aromatic residues were found to be useful in corroborating assignments, especially those due to the large shifts induced by the heme. Assignment of NOESY cross peaks was greatly facilitated by a prediction of intensities using a complete relaxation matrix analysis based on the crystal structure. These results suggest that the single-crystal X-ray structure closely resembles that of the solution structure although there is evidence that the solution structure has a more dynamic character.

Amino Acid Sequence↗
PubMed1990

Removal of a proteolytic activity associated with aggregates formed from expression of creatine kinase in Escherichia coli leads to improved recovery of active enzyme.

Expression of creatine kinase (CK) from a Torpedo californica electric organ cDNA in Escherichia coli results in an insoluble protein product with no detectable CK activity. Although this is a stable aggregate that can be isolated in an enriched form by centrifugation, initial attempts to generate enzyme activity by denaturing and refolding yielded only minute amounts of active protein. We find that these low recoveries are due to proteolysis of the CK during denaturation and refolding. While this proteolytic activity is not inhibited by either phenylmethanesulfonyl fluoride (PMSF) or EDTA, it can be largely removed from the CK aggregate by extraction with a detergent-containing buffer prior to denaturation. This treatment improves the recovery of active CK approximately 100-fold. We have also found similar proteolytic activity associated with the aggregate formed when a mutant of bovine pancreatic trypsin inhibitor (BPTI) is expressed in E. coli. Discovery of this proteolytic activity in two different expression systems suggests that it should be considered as a potential problem for recovery of active protein from other inclusion bodies as well.

Animals↗