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Biomedical subjects

M R Pincus

Publications and source records attributed to M R Pincus.

At least 109 records · Page 6Linked to original sources
PubMed1987

Conformational effects of substituting amino acids for glutamine-61 on the central transforming region of the P21 proteins.

The low-energy conformations for a series of peptides based on the sequence of the ras P21 protein from position 55 to position 67 have been computed using conformational energy analysis. These sequences differed at position 61 and contained Gln, Pro, Leu, Lys, and Arg at this position. P21 proteins with Gln, Glu, or Pro at this position do not cause cell transformation at normal levels of expression; proteins with substitutions of at least 14 other amino acids at this position (Leu, Lys, and Arg having been found in tumors in place of the normally occurring Gln-61) do cause malignant transformation of cells in culture. We find that the segments of residues 55-67 from the nontransforming proteins (Gln- or Pro-61) adopt a structure that is energetically unfavorable for the same segment with Leu, Lys, or Arg at position 61. The critical feature of this structure is an alpha-helix from residues 62 to 68. Residue 61 (Gln or Pro) adopts an extended conformation. On the other hand, the segment from transforming proteins can adopt two structures, one all alpha-helical from residue 61 to residue 68 and the other a less-regular, higher-energy structure. The segments from the normal protein can adopt the all alpha-helical structure, a finding that can explain the fact that elevated intracellular levels of the normal protein also cause cell transformation. The results of the calculations suggest that specific changes in the structure of this region can account for the oncogenic effect of the proteins in which substitutions occur.

Amino Acid Sequence↗
PubMed1985

Structural effects of substitutions on the p21 proteins.

The conformational effects of different amino acid substitutions (lysine, serine, proline, and D-valine) for glycine at position 12 in the p21 oncogene-encoded proteins have been investigated by using conformational energy calculations. The normal cellular gene codes for a glycine at position 12 in the amino acid sequence, in the middle of a hydrophobic p21-(6-15)-decapepetide from Leu-6 to Gly-15. Mutations that cause amino acid substitutions for Gly-12 result in a protein product that produces malignant transformation of cells. We now find that not only are the preferred structures for the lysine- and serine-containing peptides more restricted and more helical than those for the glycine-containing peptide, but the lowest-energy structure for each substituted peptide is exactly the same as that previously found for the peptide with Val-12, suggesting the existence of a "malignancy-causing" conformation. None of the preferred conformations for the valine-, lysine-, and serine-containing peptides contain chain reversals at positions 11 and 12. However, we find that proline, unlike these residues but like glycine, at position 12 causes helix termination at positions 11 and 12, a result that suggests that the p21 protein product with proline at position 12 may exhibit lowered transforming potential, in agreement with the results of recent genetic recombination experiments.

Amino Acid Sequence↗
PubMed1984

Experimental identification of a theoretically predicted "left-sided" binding mode for (GlcNAc)6 in the active site of lysozyme.

Using conformational energy calculations, we previously predicted that there are two distinct binding modes for hexasaccharide substrates of hen egg white lysozyme (HEWL), a "left-sided" binding mode and a "right-sided" one. The former involves such residues as Arg-45, Asn-46, and Thr-47, while the latter involves such residues as Asn-113 and Arg-114. The left-sided binding mode was predicted to predominate for (GlcNAc)6. We now present two lines of experimental evidence that indicate that left-sided binding occurs for this substrate. First, we show that ring-necked pheasant lysozyme (RNPL), in which Lys and His replace Asn and Arg at positions 113 and 114, respectively, has the same affinity for (GlcNAc)6 as does HEWL, indicating that the "right" side is not involved in equilibrium binding to the substrate. Second, we show that a monoclonal antibody, HyHEL-5, which binds specifically to an epitope including residues Arg-45, Asn-46, Thr-47, Asp-48, and Arg-68 on the far "left" side of HEWL, is competitively displaced by (GlcNAc)5 and (GlcNAc)6 but not by GlcNAc, (GlcNAc)2, or (GlcNAc)4. Only the former two substrates can bind in site F in the lower active site. Since these two substrates are the only ones that competitively displace HyHEL-5, our results suggest that the terminal saccharide residues of these substrates bind to the left side of the active site cleft, as predicted from theory.

Animals↗
PubMed1983

Correlation between the conformation of cytochrome c peptides and their stimulatory activity in a T-lymphocyte proliferation assay.

It has been shown that the minimal-length peptide having full stimulatory activity for pigeon cytochrome c-primed T cells from B10.A mice is composed of residues 88-103 of the moth (or 87-104 of the pigeon) sequence. However, to date, only residues 99-103(104)have been shown to be involved in contacting the T-cell receptor or the macrophage Ia molecule. Because the x-ray structure of tuna cytochrome c, and prior calculations on many homologous cytochrome c proteins, showed that segment 88-103(104) exists in the alpha-helical conformation, we postulate that residues 88-98 are necessary for maintaining the alpha-helical conformation of the COOH-terminal pentapeptide (99-103) involved in receptor recognition. To test this hypothesis, we have examined the conformational preferences of polypeptide segments from known antigenic regions near the carboxyl terminus of cytochrome c (pigeon, moth, and fly sequences) using conformational energy calculations for peptides in a nonpolar environment. We show here that fragments consisting of residues 88-91 and 94-98 of pigeon, moth, and fly cytochrome c have a strong alpha-helical preference, despite differences in sequence at residues 88-89 (Lys-Ala in pigeon, Ala-Asn in moth, and Pro-Asn in fly). In contrast, the tripeptide 91-93 (Arg-Ala-Asp) has a strong nonhelical preference. Furthermore, the COOH-terminal peptide 99-103 exists as a statistical coil. However, addition of residues 94-98 to residues 99-103 results in a peptide that has a strong preference for alpha-helix. From these computational results, we predict (i) that fragment 94-103, existing predominantly as an alpha-helix, should exhibit stimulatory activity and (ii) that the nonhelical peptide 91-93 can be deleted from fragment 88-103 without affecting its antigenicity. Both of these predictions have been borne out by experiments in which the two peptides were synthesized and shown to stimulate a T-cell proliferative response. These results establish a strong correlation between conformation (here, alpha-helix) and biological activity and suggest that T-cell activation is sensitive to the organized backbone structure that the antigen adopts in the nonpolar environment of the macrophage membrane or in the combining site of the T-cell receptor.

Amino Acid Sequence↗
PubMed1983

Prediction of the three-dimensional structure of the transforming region of the EJ/T24 human bladder oncogene product and its normal cellular homologue.

The three-dimensional structures of the transforming region of the product of the EJ/T24 human bladder oncogene and of the c-Ha ras-1 gene product have been calculated by using conformational energy calculations. These two genes, representing a transforming oncogene and its normal cellular homologue, encode 21,000-dalton peptides that differ by one amino acid at position 12. We therefore examined the energetically allowed conformations of the hydrophobic decapeptide surrounding this substitution site. The calculations show that the most favorable form of the c-Ha ras-1 gene product exists when glycine-12 is in a left-handed bend conformation. No other amino acid can adopt this conformation and thus the bladder oncogene peptide containing valine at position 12 has a markedly different three-dimensional structure. A simple model is proposed to account for the consequences of a position 12 mutation.

Amino Acid Sequence↗
PubMed1982

Prediction of the three-dimensional structure of the leader sequence of pre-kappa light chain, a hexadecapeptide.

The three-dimensional structure of the signal sequence for murine kappa light chain has been calculated by using conformational energy calculations. These calculations, based on tested and reliable potential energy functions, employ a novel global search technique to identify the lowest energy structures for the hexadecapeptide signal sequence, Glu-Thr-Asp-Thr-(Leu3-Trp-Val)2-Pro-Gly.l It has been found that the core hydrophobic sequence, Leu3-Trp-Val-Leu3, adopts an alpha-helical conformation that is terminated by chain reversal conformations for the four residues, Trp-Val-Pro-Gly. The amino-terminal four residues adopt low energy conformations that are fully compatible with the succeeding alpha-helix. The immediately neighboring sequence., Asp-Thr, exists in a single lowest energy double-equatorial conformation, whereas the first two residues, Glu-Thr, can adopt a variety of low energy conformations. The calculations arrive at a highly structured and specific model for the conformation of a leader sequence, compatible with recent experimental data.

Amino Acid Sequence↗
PubMed1982

Calculation of the three-dimensional structure of the membrane-bound portion of melittin from its amino acid sequence.

The structure of the NH2-terminal, 20-residue membrane-bound portion of melittin has been computed with empirical energies (ECEPP, Empirical Conformational Energy Program for Peptides). First, a search was made for long stretches of nonpolar residues. Then, the low-energy conformations of these segments were built up by combining successively the low-energy conformations of their component di- and tripeptides. The minimum-energy conformations of each of these component peptides used in this buildup process were selected so that each had a distinct backbone conformation; these distinct backbone conformations were designated as nondegenerate minima. Structured segments (i.e., those with only a few low-energy conformations) resulting from this process were identified and used as nucleation sites for building up larger structures by adding adjacent peptide segments. At each stage, the energy of each structure was minimized. Only two low-energy structures were found, both of which were alpha-helical from Gly-1 to Thr-10 and from Pro-14 to Ile-20. In one of them, the first helix continues through Thr-11-Gly-12; in the other, there is a bend at Thr-11-Gly-12. However, because of compensating changes in the dihedral angles, both structures are very similar. The calculated structures seem to agree well with experimental data.

Amino Acid Sequence↗
PubMed1981

Ligand binding to multiple equivalent sites with steric hindrance.

A general model is developed for the binding of ligands to multiple receptor sites in which steric blockage of sites is taken into account. Analytical expressions for extent of ligand binding as a function of ligand concentration are derived employing a stochastic matrix approach. Using simple numerical techniques to evaluate these expressions it is possible to obtain the inherent affinity of each site for a ligand, the number of sites available and the number of sites excluded when a ligand binds to any site. The expressions derived here are contrasted with other expressions based on simple equilibrium considerations in which there are no interactions between sites and no interactions between ligands in different sites. It is shown that the expressions derived here predict significant departures from linearity in Scatchard plots and a strong negative cooperatively, especially toward the saturation limit.

Binding Sites↗
PubMed1981

General quantitative treatment for the binding of divalent antibodies to antigens immobilized on a solid phase.

A general expression for the binding isotherm of divalent antibodies to antigens bound to solid lattices with excluded volume effects is derived by using a statistical mechanical approach. The method is based on matrix generation of the partition function for the system, from which the degree of saturation of the antigen lattice may be computed directly. The saturation function has a simple form that under appropriate conditions reduces to forms derived previously for simpler cases (such as monovalent binding with and without excluded volume effects). Solution by numerical techniques of a simple secular equation for each concentration of antibody allows computation of the saturation function. Thus, the binding expression derived is easily fit to any set of binding data obtained and results in the determination of such parameters as intrinsic affinity constants, length of excluded space (and hence molecular size), and number of sites. It predicts that under conditions wherein divalent binding is favored over univalent binding, biphasic adsorption isotherms will be obtained.

Journal Article↗
PubMed1981

Disseminated cryptococcosis in an asymptomatic alcoholic man.

Widely disseminated cryptococcosis was found on autopsy in a 50-year-old alcoholic man. The spleen, lungs, CNS, liver, kidney, and lymph nodes were all involved. In his clinical course, the patient showed no signs of immunologic anergy. His terminal hospital course resulted directly from end-stage liver disease and renal failure. The cryptococcal infection was of the nongranulomatous, diffuse type with little or no inflammatory response, which probably explains the lack of symptoms.

Antigens, Fungal↗
PubMed1977

Structures of enzyme-substrate complexes of lysozyme.

Conformational energy calculations were used to determine the binding structures of two oligosaccharides (GlcNAc)(n), in which n = 5 and 6, in the rigid active site of lysozyme (mucopeptide N-acetylmuramoylhydrolase, EC 3.2.1.17). Starting with the lowest energy binding structures of (GlcNAc)(4) as determined in a previous publication, we added a fifth GlcNAc residue to this tetramer in three different conformations, corresponding to the left-handed and right-handed helical structures and an intermediate structure, and the energy of each complex was minimized. The most stable binding conformation of the fifth residue of the pentamer was closest to the left-handed helical one. During energy minimization, the fourth residue of the pentamer moved from its initial position near the surface of the active site farther into the active site cleft at binding site D. Binding structures of (GlcNAc)(6) were then examined by addition of a residue to the lowest energy structure of (GlcNAc)(5), and it was found that the sixth residue of the hexamer binds in a conformation again close to the left-handed helical one. Stable binding regions of the rigid active site for the fifth and sixth residues were found to be near arginyl 45 and asparaginyl 46, on the opposite side of the active site cleft from arginyl 114. When the calculated structure of the lysozyme-(GlcNAc)(4) complex (used here as the starting structure for addition of the fifth and sixth residues) is compared with recent experimental data, it is found that the calculated structure is a reasonable one. Of all binding regions available to the saccharide residues, the C site binds GlcNAc with the lowest energy, in agreement with experiments.

Acetylglucosamine↗