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L M Gierasch

Publications and source records attributed to L M Gierasch.

At least 73 records · Page 4Linked to original sources
PubMed1991

Sequence requirement for peptide recognition by rat brain p21ras protein farnesyltransferase.

We tested 42 tetrapeptides for their ability to bind to the rat brain p21ras protein farnesyltransferase as estimated by their ability to compete with p21Ha-ras in a farnesyltransfer assay. Peptides with the highest affinity had the structure Cys-A1-A2-X, where positions A1 and A2 are occupied by aliphatic amino acids and position X is occupied by a COOH-terminal methionine, serine, or phenylalanine. Charged residues reduced affinity slightly at the A1 position and much more drastically at the A2 and X positions. Effective inhibitors included tetrapeptides corresponding to the COOH termini of all animal cell proteins known to be farnesylated. In contrast, the tetrapeptide Cys-Ala-Ile-Leu (CAIL), which corresponds to the COOH termini of several neural guanine nucleotide binding (G) protein gamma subunits, did not compete in the farnesyl-transfer assay. Inasmuch as several of these proteins are geranylgeranylated, the data suggest that the two isoprenes (farnesyl and geranylgeranyl) are transferred by different enzymes. A biotinylated heptapeptide corresponding to the COOH terminus of p21Ki-rasB was farnesylated, suggesting that at least some of the peptides serve as substrates for the transferase. The data are consistent with a model in which a hydrophobic pocket in the protein farnesyltransferase recognizes tetrapeptides through interactions with the cysteine and the last two amino acids.

Alkyl and Aryl Transferases↗
PubMed1991

Side chain-backbone hydrogen bonding contributes to helix stability in peptides derived from an alpha-helical region of carboxypeptidase A.

Recently, Presta and Rose proposed that a necessary condition for helix formation is the presence of residues at the N- and C-termini (called NTBs and CTBs) whose side chains can form hydrogen bonds with the initial four amides and the last four carbonyls of the helix, which otherwise lack intrahelical hydrogen bonding partners. We have tested this hypothesis by conformational analysis by circular dichroism (CD) of a synthetic peptide corresponding to a region (171-188) of the protein carboxypeptidase A; in the protein, residues 174 to 186 are helical and are flanked by NTBs and CTBs. Since helix formation in this peptide may also be stabilized by electrostatic interactions, we have compared the helical content of the native peptide with that of several modified peptides designed to enable dissection of different contributions to helix stability. As expected, helix dipole interactions appear to contribute substantially, but we conclude that hydrogen bonding interactions as proposed by Presta and Rose also stabilize helix formation. To assist in comparison of different peptides, we have introduced two concentration-independent CD parameters which are sensitive probes of helix formation.

Amino Acid Sequence↗
PubMed1991

Recognition of nascent polypeptides for targeting and folding.

A major difference between the refolding of proteins in vitro and the in vivo folding process, in which we include localization and assembly, is the need for additional factors in vivo, apart from the protein product itself. Thus, the amino acid sequence of a naturally selected protein contains not only the information specifying its three-dimensional structure, but also the information that enables these factors to recognize the nascent polypeptide. In this review, we consider how this latter information may be encoded and, in turn, interpreted by binding species.

Bacterial Proteins↗
PubMed1990

Reduced tendency to form a beta turn in peptides from the P22 tailspike protein correlates with a temperature-sensitive folding defect.

A family of mutants of the P22 bacteriophage tailspike protein has been characterized as temperature sensitive for folding (tsf) by King and co-workers [King, J. (1986) Bio/Technology 4, 297-303]. There is substantial evidence that the tsf mutations alter the folding pathway but not the stability of the final folded protein. Several point mutations are known to cause the tsf phenotype; most of these occur in regions of the tailspike sequence likely to take up reverse turns. Hence, it has been hypothesized that the correct folding of the P22 tailspike protein requires formation of turns and that the mutations causing tsf phenotypes interfere at this critical stage. We have tested this hypothesis by study of isolated peptides corresponding to a region of the P22 tailspike harboring a tsf mutation. Comparison of the tendencies of wild-type and tsf sequences to adopt turn conformations was achieved by the synthesis of peptides with flanking cysteine residues and the use of a thiol-disulfide exchange assay. We find that the wild-type sequence, either as a decapeptide (Ac-CVKFPGIETC-CONH2) or as a dodecapeptide (Ac-CYVKFPGIETLC-CONH2), has a 3-5-fold greater tendency for its termini to approach closely enough to form the intramolecular disulfide than do the peptide sequences corresponding to the tsf mutant sequences, which have a Gly----Arg substitution (Ac-CVKFPRIETC-CONH2 or Ac-CYVKFPRIETLC-CONH2). A peptide with a D-Arg substituted for the Gly has a slightly higher turn propensity than does the wild type. Together with data from nuclear magnetic resonance analysis of the oxidized peptides, this suggests that a type II beta turn is favored by the wild-type sequence. Our results on isolated peptides from the P22 tailspike protein support the model for its folding that includes reverse turn formation as a critical step.

Amino Acid Sequence↗
PubMed1990

Comparison of helix stability in wild-type and mutant LamB signal sequences.

Previous studies of isolated peptides corresponding to the wild-type signal sequence of the LamB protein of Escherichia coli and to several export-impaired mutants demonstrated that a high tendency to adopt an alpha-helical conformation in low dielectric environments was a property of functional sequences. We have now used nuclear magnetic resonance to establish further characteristics of the helical conformation of these signal peptides in a solvent mixture (50% trifluoroethanol, by volume, in water) which mimics the conformational distribution of these peptides in lipid vesicles. The interactions of signal sequences in vivo may depend on the location of the helix in the sequence, on the length of the helical segment, and on the stability of the helix. We find that the hydrophobic core has the most persistent helix conformation and that the stability of this helix correlates with in vivo function of different mutants of the LamB signal sequence. In the family of signal peptides studied here, the length of the helix required for function appears to be less rigidly restricted since a signal peptide from a functional pseudorevertant with 4 residues deleted from the hydrophobic core takes up helix as stably as wild type but incorporates fewer residues in the helix.

Amino Acid Sequence↗
PubMed1990

Cyclic pentapeptides as models for reverse turns: determination of the equilibrium distribution between type I and type II conformations of Pro-Asn and Pro-Ala beta-turns.

Cyclic pentapeptides are excellent models for reverse turns and have been used extensively in our laboratory to explore the influence of different amino acid sequences on turn preference. This paper is divided into two parts: In the first, we review our previous studies of cyclic pentapeptides. We summarize work that demonstrates the range of conformations possible within the cyclic pentapeptide backbone, the importance of sequence chirality in determining the backbone fold, and the utility of these cyclic pentapeptides as models for various turns. In the second, we present new results on two cyclic pentapeptides that contain beta-turns with Pro-Ala or Pro-Asn sequences in the i + 1 and i + 2 positions. By stereochemical criteria, a type I beta-turn is expected to be preferred by such L-L sequences. On the other hand, in proteins Asn occurs frequently in the i + 2 position of type II turns. We asked whether the same propensity would be manifest in an isolated model peptide, and if so, what the interactions were that influenced the relative stability of the type I and type II turns. To address these questions we have compared the conformational behavior of two peptides: cyclo(Gly-Pro-Ala-D-Phe-Pro) and cyclo(D-Ala-Pro-Asn-Gly-Pro). From previous studies, we anticipated that both peptides would contain an inverse gamma-turn and a beta-turn which consisted of either Gly-Pro-Ala-D-Phe or D-Ala-Pro-Asn-Gly in positions i to i + 3, respectively. Nuclear magnetic resonance analysis confirms this overall backbone conformation. Furthermore, quantitative nuclear Overhauser effect measurements in combination with molecular dynamics simulations and torsionally-forced energy minimizations have enabled us to determine that both type I and type II beta-turns are present in equilibrium in these peptides. The introduction of Asn in position i + 2 shifts this equilibrium significantly towards type II. We have done preliminary assessment of the possible side-chain/backbone conformations that contribute to the shift in populations.

Amino Acid Sequence↗
PubMed1990

Design of biologically active, conformationally constrained GnRH antagonists.

The introduction of conformational constraints into a flexible peptide hormone can be exploited to develop models for the conformation required for receptor binding and activity. In this review, we illustrate this approach to analog design using our work on antagonists of gonadotropin-releasing hormone (GnRH). Design of a conformationally constrained, competitive antagonist of GnRH, cyclo[delta 3,4 Pro-D4ClPhe-DTrp-Ser-Tyr-DTrp-NMeLeu-Arg-Pro-bet a Ala] led to the prediction of its bioactive conformation. Template forcing experiments show that this conformation is accessible to other active GnRH analogs. Two-dimensional NMR studies verified the predicted conformation in solution. The predicted binding conformation has recently been used to design two new analogs incorporating side chain-side chain linkages suggested by the conformational model: Ac-delta 3,4Pro-D4FPhe-DTrp-Dap-Tyr-DTrp-Leu-Arg-Asp-Gly- NH2 and Ac-delta 3,4Pro-D4FPhe-DTrp-Dap-Tyr-D2Nal-Leu-Arg-Pro-Asp -NH2. These analogs were synthesized and the one predicted to be most similar to the parent conformation had equivalent potency while the second, designed to refine the conformational hypothesis, was found to exhibit enhanced potency, thus confirming the original binding conformation hypothesis. These compounds and their derivatives now provide a new class of GnRH antagonists possessing both high biological potency and limited conformational flexibility, thus making them ideal for both biophysical and structure-activity studies.

Amino Acid Sequence↗
PubMed1990

Biophysical studies of signal peptides: implications for signal sequence functions and the involvement of lipid in protein export.

This review discusses efforts to understand the mode of action of signal sequences by biophysical study of synthetic peptides corresponding to these protein localization signals. On the basis of reports from several laboratories, it is now clear that signal peptides may adopt a variety of conformations, depending on their local environment. In membrane-mimetic systems like detergent micelles or lipid vesicles, they have a high tendency to form alpha helices. Ability to take up a helical conformation appears to be required at some point in the function of a signal sequence, since some peptides corresponding to export-defective signal sequences display reduced helical potential. By contrast, functional signal sequences share a high capacity to adopt alpha helices. High affinity for organized lipid assemblies, like monolayers or vesicles, is also a property of functional signal sequences. This correlation suggests a role for direct interaction of signal sequences with the lipids of the cytoplasmic membrane in vivo. Supporting this role are studies of the influence of signal peptides on lipid structure, which reveal an ability of these peptides to perturb lipid packing and to alter the phase state of the lipids. Insertion of the signal sequence in vivo could substantially reduce the barrier for translocation of the mature chain. Lastly, synthetic signal peptides have been added to native membranes and found to inhibit translocation of precursor proteins. This approach bridges the biophysical and the biochemical aspects of protein export and promises to shed light on the functional correlates of the properties and interactions observed in model systems.

Amino Acid Sequence↗
PubMed1990

Synthesis and crystal structures of Boc-L-Asn-L-Pro-OBzl.CH3OH and dehydration side product, Boc-beta-cyano-L-alanine-L-Pro-OBzl.

Boc-L-Asn-L-Pro-OBzl: C21H29O6N3.CH3OH, Mr = 419.48 + CH3 OH, monoclinic, P2(1), a = 10.049(1), b = 10.399(2), c = 11.702(1) A, beta = 92.50(1)degrees, V = 1221.7(3) A3, dx = 1.14 g.cm-3, Z = 2, CuK alpha (lambda = 1.54178 A), F(000) = 484 (with solvent), 23 degrees, unique reflections (I greater than 3 sigma(I)) = 1745, R = 0.043, Rw = 0.062, S = 1.66. Boc-beta-cyano-L-alanine-L-Pro-OBzl: C21H27O5N3, Mr = 401.46, orthorhombic, P2(1)2(1)2(1), a = 15.741(3), b = 21.060(3), c = 6.496(3) A, V = 2153(1) A3, dx = 1.24 g.cm-3, Z = 4, CuK alpha (lambda = 1.54178 A), F(000) = 856, 23 degrees, unique reflections (I greater than 3 sigma(I)) = 1573, R = 0.055, Rw = 0.078, S = 1.86. The tert.-butyloxycarbonyl (Boc) protected dipeptide benzyl ester (OBzl), Boc-L-Asn-L-Pro-OBzl, prepared from a mixed anhydride reaction using isobutylchloroformate, Boc-L-asparagine, and HCl.L-proline-OBzl, crystallized with one methanol per asymmetric unit in an extended conformation with the Asn-Pro peptide bond trans. Intermolecular hydrogen bonding occurs between the methanol and the Asn side chain and between the peptide backbone and the Asn side chain. A minor impurity due to the dehydration of the Asn side chain to a beta-CNala crystallized with a similar extended conformation and a single intermolecular hydrogen bond.

Amino Acid Sequence↗
PubMed1989

Helix formation and stability in a signal sequence.

A detailed nuclear magnetic resonance analysis of the isolated LamB signal peptide (MMITLRKLPLAVAVAAGVMSAQAMA) under conditions defined by circular dichroism spectra to mimic the conformational distribution of this peptide in membranelike environments has provided a description of specific residue conformational preferences. This 25-residue long peptide in 20 mol % trifluoroethanol in water is in dynamic equilibrium between a helical and a more random conformation, and this equilibrium is shifted toward the more random structure as the temperature is raised. Part of the molecule, residues 10-18, exists in a stable helix at all temperatures studied (5, 25, and 50 degrees C). Propagation of the helix through the C-terminal end occurs at 25 degrees C, while the temperature must be lowered to 5 degrees C to observe any significant population of a helical conformation in the N-terminal region. These results argue that the Pro and Gly residues, which flank the helical segment, act to disfavor helix propagation on their N- or C-terminal sides, respectively. The influence of the Pro residue is stronger than that of the Gly. Furthermore, the most stable part of the helix in this signal peptide under the conditions studied is the hydrophobic core, which is the hallmark of functional signal sequences.

Amino Acid Sequence↗
PubMed1989

Functional and nonfunctional LamB signal sequences can be distinguished by their biophysical properties.

The role of the signal sequence in the secretion of proteins remain unclear despite extensive research. We have examined properties of synthetic peptides corresponding to a family of signal sequences derived from the lamB gene of Escherichia coli, including five examples of known phenotype that contain mutations in the signal sequence. By circular dichroism spectroscopy, the wild type and export-component mutant signal peptides show a high alpha-helix content in membrane mimetic environments (sodium dodecyl sulfate micelles and phospholipid vesicles). Tendency to adopt helical conformations is clearly not sufficient to define a functional signal sequence, however, as some nonfunctional mutant signal peptides also contain a relatively high proportion of alpha-helix. The affinity of these peptides for phospholipid monolayers as assessed by surface tensiometry reveals further distinguishing properties. Export-competent peptides show an increased affinity for and greater perturbation of phospholipid monolayers and bilayers than to export-defective peptides. These results suggest a lipid binding role for the signal sequence during protein export in addition to its recognition by proteins of the export pathway.

Amino Acid Sequence↗
PubMed1989

Conformations and orientations of a signal peptide interacting with phospholipid monolayers.

The interaction of a chemically synthesized 25-residue signal peptide of LamB protein from Escherichia coli with phospholipids has been studied with a film balance technique. The conformation, orientation, and concentration of the peptides in lipid monolayers have been determined from polarized infrared spectroscopy, ultraviolet spectroscopy, and assay of 14C-labeled peptide in transferred films. When the LamB signal peptide is injected into the subphase under a phosphatidylethanolamine-phosphatidylglycerol monolayer at low initial pressure, insertion of a portion of the peptide into the lipid film is evidenced by a rapid rise in film pressure. Spectroscopic results obtained on films transferred to quartz plates and Ge crystals show that the peptide is a mixture of alpha-helix and beta-conformation where the long axis of the alpha-helix penetrates the monolayer plane and the beta-structure is coplanar with the film. By contrast, when peptide is injected under lipid at high initial pressure, no pressure rise is observed, and the spectroscopic results show the presence of only beta-structure which is coplanar with the monolayer. The spectroscopic and radioassay results are all consistent with the picture of a peptide anchored to the monolayer through electrostatic binding with a helical portion inserted into the lipid region of the monolayer and a beta-structure portion resident in the aqueous phase. The negative charges on the lipid molecules are roughly neutralized by the positive charges of the peptide.

Bacterial Outer Membrane Proteins↗
PubMed1989

Signal sequences.

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Amino Acid Sequence↗
PubMed1987

Nuclear magnetic resonance analysis and conformational characterization of a cyclic decapeptide antagonist of gonadotropin-releasing hormone.

Two-dimensional proton nuclear magnetic resonance spectroscopy at 500 MHz has been carried out on the cyclic decapeptide antagonist of gonadotropin-releasing hormone: cyclo-(delta 3-Pro1-D-pClPhe2-D-Trp3-Ser4-Tyr5-D-Trp6-NMeLeu7-Arg8- Pro9-beta-Ala 10). The antagonist exists in two slowly interconverting conformations. All data are consistent with the conclusion that one form has all-trans peptide bonds and the other has a cis beta-Ala10-delta3-Pro1 bond. With the use of sequential assignment methods, chemical shift assignments were obtained for all backbone and side-chain protons of both conformational isomers except for the serine and tyrosine hydroxyl groups and the C gamma, C delta, and guanidinium group protons of the arginine. Temperature dependence of spectral parameters and magnitudes of observed nuclear Overhauser effects support the interpretation that both conformers of the antagonist consist of two beta-turns (type II', D-Trp6-NMeLeu7; type II, delta 3-Pro1-D-pClPhe2) connected by extended antiparallel beta-like strands.

Gonadotropin-Releasing Hormone↗
PubMed1987

Protein translocation into Escherichia coli membrane vesicles is inhibited by functional synthetic signal peptides.

A synthetic peptide corresponding to the signal sequence of wild type Escherichia coli lambda-receptor protein (LamB) inhibits in vitro translocation of precursors of both alkaline phosphatase and outer membrane protein A into E. coli membrane vesicles (half-maximal inhibition at 1-2 microM). By contrast, the inhibitory effect was nearly absent in a synthetic peptide corresponding to the signal sequence from a mutant strain that harbors a deletion mutation in the LamB signal region and displays an export-defective phenotype for this protein in vivo. Two peptides derived from pseudorevertant strains that arose from the deletion mutant and exported LamB in vivo were found to inhibit in vitro translocation with effectiveness that correlated with their in vivo export ability. Controls indicated that these synthetic signal peptides did not disrupt the E. coli membrane vesicles. These results can be interpreted to indicate that the presequences of exported proteins interact specifically with a receptor either in the E. coli inner membrane or in the cytoplasmic fraction. However, biophysical data for the family of signal peptides studied here reveal that they will spontaneously insert into a lipid membrane at concentrations comparable to those that cause inhibition. Hence, an indirect effect mediated by the lipid bilayer of the membrane must be considered.

Alkaline Phosphatase↗
PubMed1986

Conformations of signal peptides induced by lipids suggest initial steps in protein export.

Despite the requirement for a functional signal sequence in protein export, little is known of the conformational properties and membrane interactions of these highly hydrophobic amino terminal extensions on nearly all exported proteins. The Escherichia coli lambda phage receptor signal sequence was studied in phospholipid monolayers by circular dichroism and Fourier transform infrared spectroscopy; the signal peptide was shown to prefer an alpha-helical conformation when inserted into the lipid phase. However, interaction with the lipid surface without insertion induced the signal sequence, which is unstructured in bulk aqueous solution, to adopt a beta structure. These observations are combined in a model for the initial steps in signal sequence-membrane interaction in vivo.

Bacteriophage lambda↗
PubMed1986

Dynamics of a hydrophobic peptide in membrane bilayers by solid-state nuclear magnetic resonance.

Solid-state NMR studies of the dynamics of a synthetic hydrophobic peptide, tert-butyloxycarbonylleucylphenylalanine methyl ester (Boc-Leu-Phe-OMe), in phospholipid bilayers are described. Motionally averaged powder pattern line shapes from 2H- and 15N-labeled backbone and side-chain sites of the peptide in phospholipid bilayers demonstrate the presence of both overall and internal reorientations of substantial amplitude. The peptide motions are shown to be strongly influenced by the motions of the lipids.

Dimyristoylphosphatidylcholine↗