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Biomedical subjects

M R Pincus

Publications and source records attributed to M R Pincus.

At least 55 records · Page 3Linked to original sources
PubMed1994

Conformational effects of environmentally induced, cancer-related mutations in the p53 protein.

The tumor suppressor gene p53 has been identified as the most frequent target of genetic alterations in human cancers. A considerable number of environmentally induced, cancer-related p53 mutations in human tumors have been found in a highly conserved proline-rich sequence of the p53 protein encompassed by amino acid residues 147-158. Using conformational energy analysis based on ECEPP (Empirical Conformational Energy for Peptides Program), we have determined the low-energy three-dimensional structures for this dodecapeptide sequence for the human wild-type p53 protein and three environmentally induced, cancer-related mutant p53 proteins with His-151, Ser-152, and Val-154, respectively. The results suggest that the wild-type sequence adopts a well-defined low-energy conformation and that the mutant peptides adopt well-defined conformations that are distinctly different from the conformation of the wild-type peptide. These results are consistent with experimental conformational studies demonstrating altered detectability of antigenic epitopes in wild-type and mutant p53 proteins. These results suggest that the oncogenic effects of these environmentally induced, cancer-related, mutant p53 proteins may be mediated by distinct local conformational changes in the protein.

Amino Acid Sequence↗
PubMed1994

Interaction between cytochrome P450 2B1 and cytochrome bs: inhibition by synthetic peptides indicates a role for P450 residues Lys-122 and Arg-125.

Binding of cytochrome b5 to rat cytochrome P450 2B1 was inhibited (by 75%) by a synthetic peptide corresponding to P450 residues 116-134. The role of Lys-122 and Arg-125 were evaluated using peptides in which one or both of these basic residues were replaced with Glu. The Lys-122 substitution nearly abolished while the Arg-125 replacement decreased (by 20%) the inhibitory potential of the peptide. Substitution of both residues resulted in a peptide with no inhibitory activity. These results thus indicate a role for a specific P450 region as well as two basic residues within this region in the cytochrome P450-cytochrome b5 interaction.

Amino Acid Sequence↗
PubMed1994

Specificity of the cytochrome P-450 interaction with cytochrome b5.

The specificity of the interaction of cytochrome b5 with different forms of cytochrome P-450 was examined. Immunopurification of cytochromes P-450 1A1, 2B1 and 2E1 from rat liver microsomes resulted in co-purification of cytochrome b5 with cytochrome P-450 forms 2B1 and 2E1 but not 1A1. This specificity was evaluated in conjunction with multiple sequence alignment of the three cytochrome P-450s and a molecular model of the cytochrome P-450-cytochrome b5 complex [(1989) Biochemistry 28, 8201-8205]. These analyses suggest two basic residues in the arginine cluster region of P-450, which are present in P-450s 2B1 and 2E1 but are absent in P-450 1A1, as potential binding sites for cytochrome b5.

Amino Acid Sequence↗
PubMed1994

Conformation of the transmembrane domain of the epidermal growth factor receptor.

The transmembrane domain of growth factor receptors, such as the epidermal growth factor receptor (EGFR) and the related c-erbB-2/neu oncogene protein, has been implicated in the process of receptor dimerization and mitogenic signal transduction, and hence in cellular transformation and oncogenesis. Amino acid substitutions in the transmembrane domain of the c-erbB-2/neu protein that cause a transforming effect may exert this effect through a conformational change from a bend conformation to an alpha-helical structure in this region of the protein, but similar amino acid substitutions at homologous positions in the transmembrane domain of the EGFR (e.g., Val-->Glu at position 627) fail to have a transforming effect. To examine whether this failure may be due to structural effects, we have used conformational energy analysis to determine the preferred three-dimensional structures for the nonapeptide sequence of the transmembrane domain of the EGFR from residues 623-631 with Val or Glu at position 627. The global minimum energy conformations of both nonapeptides were found to be non-alpha-helical with bends at positions 624-625 and 627-628. The failure of the Val-->Glu substitution to produce a conformational change to an alpha-helix in this region may be responsible for its lack of transforming effect. However, the presence of higher energy alpha-helical conformations for the nonapeptide from the normal EGFR may provide an explanation for the presence of a transforming effect from overexpression of the EGFR.

Amino Acid Sequence↗
PubMed1994

Comparison of the low energy conformations of an oncogenic and a non-oncogenic p21 protein, neither of which binds GTP or GDP.

Oncogenic p21 protein, encoded by the ras-oncogene, that causes malignant transformation of normal cells and many human tumors, is almost identical in sequence to its normal protooncogene-encoded counterpart protein, except for the substitution of arbitrary amino acids for the normally occurring amino acids at critical positions such as Gly 12 and Gln 61. Since p21 is normally activated by the binding of GTP in place of GDP, it has been postulated that oncogenic forms must retain bound GTP for prolonged time periods. However, two multiply substituted p21 proteins have been cloned, neither of which binds GDP or GTP. One of these mutant proteins with Val for Gly 10, Arg for Gly 12, and Thr for Ala 59 causes cell transformation, while the other, similar protein with Gly 10, Arg 12, Val for Gly 13 and Thr 59 does not transform cells. To define the critical conformational changes that occur in the p21 protein that cause it to become oncogenic, we have calculated the low energy conformations of the two multiply substituted mutant p21 proteins using a new adaptation of the electrostatically driven Monte Carlo (EDMC) technique, based on the program ECEPP. We have used this method to explore the conformational space available to both proteins and to compute the average structures for both using statistical mechanical averaging. Comparison of the average structures allows us to detect the major differences in conformation between the two proteins. Starting structures for each protein were calculated using the recently deposited x-ray crystal coordinates for the p21 protein, that was energy-refined using ECEPP, and then perturbed using the EDMC method to compute its average structure. The specific amino acid substitutions for both proteins were then generated into the lowest energy structure generated by this procedure, subjected to energy minimization and then to full EDMC perturbations. We find that both mutant proteins exhibit major differences in conformation in specific regions, viz., residues 35-47, 55-78, 81-93, 96-110, 115-126, and 123-134, compared with the EDMC-refined x-ray structure of the wild-type protein. These regions have been found to be the most flexible in the p21 protein bound to GDP from prior molecular dynamics calculations (Dykes et al., 1993). Comparison of the EDMC-average structure of the transforming mutant with that of the nontransforming mutant reveals major structural differences at residues 10-16, 32-40, and 60-68. These structural differences appear to be the ones that are critical in activation of the p21 protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystallography, X-Ray↗
PubMed1994

Prediction of conformation of rat galanin in the presence and absence of water with the use of Monte Carlo methods and the ECEPP/3 force field.

The conformation of the 29-residue rat galanin neuropeptide was studied using the Monte Carlo with energy minimization (MCM) and electrostatically driven Monte Carlo (EDMC) methods. According to a previously elaborated procedure, the polypeptide chain was first treated in a united-residue approximation, in order to enable extensive exploration of the conformational space to be carried out (with the use of MCM). Then the low-energy united-residue conformations were converted to the all-atom representations, and EDMC simulations were carried out for the all-atom polypeptide chains, using the ECEPP/3 force field with hydration included. In order to estimate the effect of environment on galanin conformation, the low-energy conformations obtained as a result of these simulations were taken as starting structures for further EDMC runs that did not include hydration. The lowest-energy conformation obtained in aqueous solution calculations had a nonhelical N-terminal part packed against the nonpolar face of a residual helix that extended from Pro13 toward the C-terminus. One next lowest-energy structure was a nearly-all-helical conformation, but with a markedly higher energy. In contrast, all of the low-energy conformations in the absence of water were all-helical differing only by the extent to which the helix was kinked around Pro13. These results are in qualitative agreement with the available NMR and CD data of galanin in aqueous and nonaqueous solvents.

Amino Acid Sequence↗
PubMed1994

The c-erbB-2 protein in oncogenesis: molecular structure to molecular epidemiology.

The c-erbB-2 (HER-2, neu) oncogene has been implicated frequently in many human tumors. This oncogene codes for a 185-kDa protein that functions as a transmembrane growth factor receptor. Overexpression of the normal protein or point mutations in the transmembrane domain of the protein have been shown to have a transforming effect. Molecular structure studies of the transmembrane domain provide a plausible explanation for this transforming effect in both cases and relate this to the process of receptor dimerization in the membrane, degradation of the protein with release of the extracellular domain (ECD) into the extracellular environment, and aberrant signal transduction. The release of the ECD into the extracellular environment provides a potential biomarker for the study of signal transduction at the molecular level in vivo. The ECD can be quantitated immunologically in the serum of individuals with cancers associated with p185 overexpression and in individuals at risk for the development of such cancers and can be used to distinguish these individuals from normal, healthy controls. Identification of such individuals by their serum ECD levels combined with specific chemotherapeutic/chemoprophylactic interventions could allow for improvement treatment and prevention of c-erbB-2-related cancers.

Amino Acid Sequence↗
PubMed1993

Unfolding and refolding of the native structure of bovine pancreatic trypsin inhibitor studied by computer simulations.

A new procedure for studying the folding and unfolding of proteins, with an application to bovine pancreatic trypsin inhibitor (BPTI), is reported. The unfolding and refolding of the native structure of the protein are characterized by the dimensions of the protein, expressed in terms of the three principal radii of the structure considered as an ellipsoid. A dynamic equation, describing the variations of the principal radii on the unfolding path, and a numerical procedure to solve this equation are proposed. Expanded and distorted conformations are refolded to the native structure by a dimensional-constraint energy minimization procedure. A unique and reproducible unfolding pathway for an intermediate of BPTI lacking the [30,51] disulfide bond is obtained. The resulting unfolded conformations are extended; they contain near-native local structure, but their longest principal radii are more than 2.5 times greater than that of the native structure. The most interesting finding is that the majority of expanded conformations, generated under various conditions, can be refolded closely to the native structure, as measured by the correct overall chain fold, by the rms deviations from the native structure of only 1.9-3.1 A, and by the energy differences of about 10 kcal/mol from the native structure. Introduction of the [30,51] disulfide bond at this stage, followed by minimization, improves the closeness of the refolded structures to the native structure, reducing the rms deviations to 0.9-2.0 A. The unique refolding of these expanded structures over such a large conformational space implies that the folding is strongly dictated by the interactions in the amino acid sequence of BPTI. The simulations indicate that, under conditions that favor a compact structure as mimicked by the volume constraints in our algorithm, the expanded conformations have a strong tendency to move toward the native structure; therefore, they probably would be favorable folding intermediates. The results presented here support a general model for protein folding, i.e., progressive formation of partially folded structural units, followed by collapse to the compact native structure. The general applicability of the procedure is also discussed.

Animals↗
PubMed1993

A comparison of techniques for introducing macromolecules into living cells.

Flow cytometry has been used to evaluate several techniques for introducing macromolecules into large numbers of living cells. One technique is cell fusion with red blood cell ghosts loaded with a fluorescent reporter molecule (RBCF). The second technique, termed osmotic lysis of pinosomes (OLP), involves a brief exposure of cells to a hypertonic solution containing the reporter molecule; subsequently, a hypotonic media is added which lyses the pinosomes formed during the hypertonic treatment. A third technique, scrape loading (SL), involves the creation of transient holes in the cell membrane through the application of mechanical forces, which allows for the passage of reporter molecules into cells. A comparison of these techniques is presented here. OLP appears to offer several advantages: It is a simple procedure, virtually all cells are fluorescently labelled, and it is capable of loading larger amounts of material more uniformly into cells while maintaining excellent viability.

Cell Fusion↗
PubMed1993

Calculation of protein backbone geometry from alpha-carbon coordinates based on peptide-group dipole alignment.

An algorithm is proposed for the conversion of a virtual-bond polypeptide chain (connected C alpha atoms) to an all-atom backbone, based on determining the most extensive hydrogen-bond network between the peptide groups of the backbone, while maintaining all of the backbone atoms in energetically feasible conformations. Hydrogen bonding is represented by aligning the peptide-group dipoles. These peptide groups are not contiguous in the amino acid sequence. The first dipoles to be aligned are those that are both sufficiently close in space to be arranged in approximately linear arrays termed dipole paths. The criteria used in the construction of dipole paths are: to assure good alignment of the greatest possible number of dipoles that are close in space; to optimize the electrostatic interactions between the dipoles that belong to different paths close in space; and to avoid locally unfavorable amino acid residue conformations. The equations for dipole alignment are solved separately for each path, and then the remaining single dipoles are aligned optimally with the electrostatic field from the dipoles that belong to the dipole-path network. A least-squares minimizer is used to keep the geometry of the alpha-carbon trace of the resulting backbone close to that of the input virtual-bond chain. This procedure is sufficient to convert the virtual-bond chain to a real chain; in applications to real systems, however, the final structure is obtained by minimizing the total ECEPP/2 (empirical conformational energy program for peptides) energy of the system, starting from the geometry resulting from the solution of the alignment equations. When applied to model alpha-helical and beta-sheet structures, the algorithm, followed by the ECEPP/2 energy minimization, resulted in an energy and backbone geometry characteristic of these alpha-helical and beta-sheet structures. Application to the alpha-carbon trace of the backbone of the crystallographic 5PTI structure of bovine pancreatic trypsin inhibitor, followed by ECEPP/2 energy minimization with C alpha-distance constraints, led to a structure with almost as low energy and root mean square deviation as the ECEPP/2 geometry analog of 5PTI, the best agreement between the crystal and reconstructed backbone being observed for the residues involved in the dipole-path network.

Algorithms↗
PubMed1993

Prediction of protein conformation on the basis of a search for compact structures: test on avian pancreatic polypeptide.

Based on the concept that hydrophobic interactions cause a polypeptide chain to adopt a compact structure, a method is proposed to predict the structure of a protein. The procedure is carried out in four stages: (1) use of a virtual-bond united-residue approximation with the side chains represented by spheres to search conformational space extensively using specially designed interactions to lead to a collapsed structure, (2) conversion of the lowest-energy virtual-bond united-residue chain to one with a real polypeptide backbone, with optimization of the hydrogen-bond network among the backbone groups, (3) perturbation of the latter structure by the electrostatically driven Monte Carlo (EDMC) procedure, and (4) conversion of the spherical representation of the side chains to real groups and perturbation of the whole molecule by the EDMC procedure using the empirical conformational energy program for peptides (ECEPP/2) energy function plus hydration. Application of this procedure to the 36-residue avian pancreatic polypeptide led to a structure that resembled the one determined by X-ray crystallography; it had an alpha-helix starting at residue 13, with the N-terminal portion of the chain in an extended conformation packed against the alpha-helix. Similar structures with slightly higher energies, but looser packing, were also obtained.

Amino Acids↗
PubMed1993

Molecular dynamics of the H-ras gene-encoded p21 protein; identification of flexible regions and possible effector domains.

We previously reported a complete computer-based three-dimensional structure for residues 1-171 of the Gly 12-containing ras-gene-encoded p21 protein complexed with GDP. This structure was subsequently shown to closely agree with a high-resolution x-ray crystallographic structure of p21. In this communication, we report a molecular dynamics stimulation of the modelled structure in an explicit shell of water molecules to identify domains within the protein that are unusually flexible. These domains represent regions which are most likely to undergo important conformational changes when the protein is activated by binding to GTP or by oncogenic amino acid substitutions such as Val for Gly 12. The starting structure was surrounded with water molecules, temperature-equilibrated and then followed over a 100 ps trajectory during which time the energy converged after about 50 ps. Regions of the protein that were found to have the largest coordinate fluctuations involved residues 12-16, 30-35, 40-52, 60-73, 85-89, 101-109, 119-123, and 127-131. Many of these sequences with high flexibility have been implicated in the functioning of this protein. Since the overall largest fluctuations were observed for residues 101-106 and 119-123, p21 peptides containing these residues (96-110 and 115-126) were synthesized and were found to inhibit strongly the effects of oncogenic p21 protein in an oocyte maturation assay. These results indicate that the flexible p21 sequences may constitute critical functional domains of the activated protein and that this general approach may be useful for identification of important functional domains in proteins.

3T3 Cells↗
PubMed1993

Activated conformations of the ras-gene-encoded p21 protein. 2. Comparison of the computed and high-resolution x-ray crystallographic structures of Gly-12 p21.

The ras-oncogene-encoded p21 protein is a G-protein that has been shown to cause the malignant transformation of normal cells and has been implicated in causing human tumors. p21 is thought to be activated by the binding of GTP in place of GDP to the protein. We have previously constructed the three-dimensional structure of the p21 protein bound to GDP from an available alpha-carbon tracing of this protein using a combination of molecular dynamics and energy minimization (Dykes, et al., J. Biomol. Struct. Dynamics, 9:1025-1044). Until the recent publication of the all-heavy-atom x-ray crystallographic molecular coordinates of p21 residues 1-166 bound to a non-hydrolyzable GTP derivative (GppNp), no all-atom structure of the p21 protein has been available in the Brookhaven National Laboratories Protein Data Bank (PDB). In this communication we compare our computed structure for the p21-GDP complex to this x-ray crystal structure. We find that the two structures agree quite closely with one another, the overall RMS deviation for the backbone being 1.47 A and 2.71 A for all of the atoms. We have identified the regions of the protein that are responsible for the most significant deviations between the two structures, i.e., residues 32-40 and 61-74. We find that the main chain in the 32-40 segment deviates significantly from residue 32 to residue 36 and the side chain phenolic rings of residue 32 differ greatly between the two structures. The 61-74 region is the least-well defined region in the whole protein crystallographically having, by far, the highest temperature factor (B-factor). The backbone and side chain conformations in the 61-74 segment differ markedly, the overall RMS deviation being 3.1 A for the backbone and 5.7 A for all atoms. Both of these regions have been found in x-ray crystallographic studies of p21-GDP and p21-GTP complexes to undergo significant changes in conformation upon the binding of GTP in place of GDP to the protein. We have further compared our computed structure of the p21 protein with the x-ray crystal structure with regard to the conformations of individual segments, in particular, structurally conserved sequences (SCR), i.e., those sequences that have structural and sequence homology to corresponding sequences in the related G-protein, bacterial elongation factor Tu(EF-Tu), and variable loop regions.(ABSTRACT TRUNCATED AT 400 WORDS)

Computer Simulation↗
PubMed1993

Structure-function correlations of calcium binding and calcium channel activities based on 3-dimensional models of human annexins I, II, III, V and VII.

The annexins are a family of calcium-dependent phospholipid-binding proteins which share a high degree of primary sequence similarity. Using a model of the crystal structure of annexin V as a template, 3-dimensional models of human annexins I, II, III and VII were constructed by homology modeling (J. Greer, J. Mol. Biol. 153, 1027-1042, 1981; J.M. Chen, G. Lee, R.B. Murphy, R.P. Carty, P.W. Brant-Rauf, E. Friedman and M.R. Pincus, J. Biomolec. Str. Dyn. 6, 859-87, 1989) for the 316 amino acid portions corresponding to the annexin V structure published by Huber et al. (J. Mol. Biol. 223, 683-704, 1992). These methods were used to study structure-function correlations for calcium ion binding and calcium channel activity. Published experimental data are specifically shown to be consistent with the annexin models. Possible intramolecular disulfide bridges were identified in annexin I (between Cys297 and Cys316) and in annexins II and VII (between Cys115 and Cys243). Each of the annexin models have 3 postulated calcium binding sites, usually via a Gly-Xxx-Gly-Thr loop with an acidic Glu or Asp residue 42 positions C-terminal to the first Gly. Despite a nonconserved binding site sequence, annexins I and II are able to coordinate calcium in domain 3 since the residue in the second loop position is directed toward the solvent away from the binding pocket. This finding also suggests a mechanism for a conformational change upon binding calcium. Highly conserved Arg and acidic sidechains stabilize the channel pore structure; annexin channels probably exist in a closed state normally. Arg271 may be involved in channel opening upon activation: basic residue 254 can stabilize Glu112, which allows Arg271 to interact with residue 95 instead of Glu112. Residue 267, found on the convex surface at the pore opening, may also be important in modifying channel activity.

Amino Acid Sequence↗
PubMed1993

A nickel-binding serpin, pNiXa, induces maturation of Xenopus oocytes and shows synergism with oncogenic ras-p21 protein.

A nickel-binding serine proteinase inhibitor, pNiXa (43 kDa), was isolated from Xenopus ovary and assayed for effects on oocyte maturation. Microinjection of pNiXa (0.12 pmol/50 nl) induced maturation in 60% of Xenopus oocytes, beginning at 4 hours and reaching completion by 9 hours. Microinjection of oncogenic ras-p21 protein (0.12 pmol/50 nl) induced maturation in 79% of oocytes, beginning at 6 hours and reaching completion by 12 hours. Microinjection of pNiXa in combination with ras-p21 protein had a synergistic effect on maturation, which occurred in 92% of oocytes, beginning at 4 hours and reaching completion by 9 hours. Oocyte maturation did not occur in control oocytes, which received a microinjection of bovine serum albumin. In oocytes exposed to a combination of pNiXa (0.12 pmol/50 nl, by microinjection) and progesterone (10 micrograms/ml, in the medium), maturation was intermediate (68% at 9 hours) between that induced by pNiXa (60%) or progesterone (85%) alone. This study shows (a) that pNiXa is a potent inducer of oocyte maturation, (b) that pNiXa's effect is synergistic with that of oncogenic ras-p21 protein, and (c) that pNiXa partially antagonizes progesterone induction of oocyte maturation.

Animals↗
PubMed1992

Effects of compact volume and chain stiffness on the conformations of native proteins.

An investigation of the statistical properties of the native conformations of proteins, observed from crystal structures, is reported. Protein conformations were analyzed in terms of a bond vector correlation function and molecular volume. It was observed that, while the volume of a protein structure varies nearly linearly with the number of residues, the bond vector correlation function exhibits a universal feature for all sizes of proteins. To interpret the nature of the bond vector correlation function of native protein structures quantitatively, Monte Carlo simulations of realistic polypeptide chains of specific but arbitrary amino acid sequence were carried out. The molecule was constrained in an ellipsoidal volume determined by its chain length, and conformations with unacceptable nonbonded contacts between different amino acid residues were excluded. The interactions within a terminally blocked single residue, which correlate two nearest-neighbor peptide groups in a chain, were taken into account by an energetically biased sampling of its phi-psi space. The simulated chain correlation functions were found to be in good agreement with those of the crystal structures of beta-sheet-type and mixed-type (alpha+beta) proteins of similar length. On the basis of these calculations, it is concluded that the observed conformations of these native proteins may arise from two basic factors: the compactness of structures under hydrophobic interactions and the intrinsic stiffness of polypeptide chains due to the interactions within each terminally blocked residue.

Biophysical Phenomena↗