Folding of a peptide corresponding to the .alpha.-helix in bovine pancreatic trypsin inhibitor

Goodman, Elizabeth M.; Kim, Peter

doi:10.1021/bi00436a033

Cited by 57 publications

(29 citation statements)

References 24 publications

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“…The opposite was true of the central 8-sheet; its structure was distorted by the surrounding protein, which diminished as the protein became more flexible. The C-terminal a-helix is indeed partially helical in solution at low temperature as measured by CD (32). Experimental work suggests that the isolated a-sheet is unstructured, however (33).…”

Section: Discussionmentioning

confidence: 99%

A model of the molten globule state from molecular dynamics simulations.

Daggett

Levitt

1992

Proc. Natl. Acad. Sci. U.S.A.

154

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It is generally accepted that a protein's primary sequence determines its three-dmenslonal structure. It has proved difficult, however, to obtain detailed structural information about the actual protein folding process and intermediate states. We present the results of molular dynamics simulations of the unfoldin of reduced bovine pancreatic trypsin inhibitor. The resulting partially "denatured" state was compact but expanded relative to the native state (11-25%); the expansion was not caused by an influx of water molecules. The structures were mobile, with overal secondary structure contents comparable to those of the native protein.The protein experienced relatively local unfolding, with the largest changes in the structure occurring in the loop regions.A hydrophobic core was maintained althongh ping of the side chains was compromised. The properties displayed in the simulation are consistent with unfolding to a molten globule state. Our smulations provide an in-depth view of this state and details of water-protein interactions that cannot yet be obtained experimentally.A protein must assume a stable and precisely ordered conformation to perform its biological function properly. Although much is known about these conformations and their synthesis, little is known about the detailed structures and dynamic transitions that occur during protein folding. Determination of the structural characteristics of partially folded intermediate states are crucial for understanding the mechanism of folding. Unfortunately, the cooperative nature of folding results in only minute amounts of these intermediates at equilibrium. So, instead, kinetic means have been used to characterize transiently populated intermediates (1-3). However, it has been shown that a few proteins adopt stable, partially folded equilibrium intermediates under unfolding conditions (4). There is some evidence that these intermediates, termed "molten globules," also occur along the kinetic pathway (ref. 4 and references therein). However, the molecular details of even the equilibrium folding intermediates remain difficult to characterize experimentally.Here we present the results of molecular dynamics (MD) simulations of protein unfolding and the resulting partially unfolded structures. The approach of studying unfolding instead of folding has computational advantages, because simulations proceed from a well-defined starting structure. In principle, MD is capable of providing detailed information about unfolding and the resulting unfolded state that cannot be obtained experimentally. This method has yielded realistic representations ofa native protein in water (5, 6) and has been useful in understanding the nature of less-structured peptides (7-9) and the unfolding-folding transitions that they undergo (7,10,11 (14). All atoms were explicitly present during the simulations, and the potential energy function and associated parameters are based on those described earlier (15, 16), except for the parameters for chloride (R* = 2.35 A; e* = 0.10 kcal/mol...

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Section: Discussionmentioning

confidence: 99%

A model of the molten globule state from molecular dynamics simulations.

Daggett

Levitt

1992

Proc. Natl. Acad. Sci. U.S.A.

154

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“…Stable cl-helices have been observed for several peptides based on native sequences, including the Cpeptide of ribonuclease A [IO,1 I], and the Po15 peptide corresponding to the a-helix of BPTI [12]. Using the RNAse A peptide as a model, Marqusee and Baldwin [ 131 designed, de nova, short peptides having as much as 80% helical content at 0°C.…”

Section: Introductionmentioning

confidence: 99%

A peptide corresponding to the N‐terminal 13 residues of T4 lysozyme forms an α‐helix

McLeish¹,

Nielsen²,

Wade³

et al. 1993

FEBS Letters

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Solid-phase methods have been used to synthesize LYS(l-13), a peptide corresponding to the first 13 residues of T4 Iysozyme. 2D 'H NMR techniques were used to investigate its solution structure in the presence of SDS micelles. The identification of numerous medium-range NOESY crosspeaks and several slowly exchanging NH protons indicated the presence of an a-helical structure. This was confirmed by simulated annealing calculations performed using XPLOR.

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“…The ellipticity of pro-wt at 222 nM decreases linearly with temperature with Amre = -27 deg cm2 dmol" "C" . This temperature dependence is typical of random polypeptides and small proteins in high Gu-HCI (Goodman & Kim, 1989;Merutka et al, 1991), indicating that the pro-wt is unfolded at all temperatures. The profile of pro-R1 shows that unfolding occurs over the temperature range of 40 to 80°C.…”

Section: Analysis Of the Temperature Unfolding Profile Circular Dichrmentioning

confidence: 76%

“…2). The mean residue ellipticity at 222 nM is -2,000 deg cm2 dmol" at 25 "C. This value is characteristic of a largely random coil structure (Goodman & Kim, 1989;Merutka et al, 1991). The mean residue ellipticity of pro-R1 at 222 nM is -5,100 deg cm2 dmol" (Fig.…”

Section: Characterization Of the Consensus Sequencementioning

confidence: 91%

Stabilizing the subtilisin BPN' pro‐domain by phage display selection: How restrictive is the amino acid code for maximum protein stability?

et al. 1998

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We have devised a procedure using monovalent phage display to select for stable mutants in the pro-domain of the serine protease, subtilisin BPN'. In complex with subtilisin, the pro-domain assumes a compact structure with a four-stranded antiparallel P-sheet and two three-turn a-helices. When isolated, however, the pro-domain is 97% unfolded. These experiments use combinatorial mutagenesis to select for stabilizing amino acid combinations at a particular structural locus and determine how many combinations are close to the maximum protein stability. The selection for stability is based on the fact that the independent stability of the pro-domain is very low and that binding to subtilisin is thermodynamically linked to folding. Two libraries of mutant pro-domains were constructed and analyzed to determine how many combinations of amino acids at a particular structural locus result in the maximum stability. A library comprises all combinations of four amino acids at a structural locus. Previous studies using combinatorial genetics have shown that many different combinations of amino acids can be accommodated in a selected locus without destroying function. The present results indicate that the number of sequence combinations at a structural locus, which are close to the maximum stability, is small. The most striking example is a selection at an interior locus of the pro-domain. After two rounds of phagemid selection, one amino acid combination is found in 40% of sequenced mutants. The most frequently selected mutant has a AGunfolding = 4 kcal/mol at 25 "C, an increase of 6 kcal/mol relative to the naturally occumng sequence. Some implications of these results on the amount of sequence information needed to specify a unique tertiary fold are discussed. Apart from possible implications on the folding code, the phage display selection described here should be useful in optimizing the stability of other proteins, which can be displayed on the phage surface.Keywords: combinatorial genetics; protein folding; site-directed mutagenesis; stopped flow kinetics; thermodynamics Recent studies have employed combinatorial genetics to investigate how amino acid sequence encodes unique tertiary folds (Sauer, 1996). Many of these studies have shown that a high degree of degeneracy in amino acid sequences is compatible with a particular backbone topology. A simple binary code of polar and apolar Reprint requests to:

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Folding of a peptide corresponding to the .alpha.-helix in bovine pancreatic trypsin inhibitor

Cited by 57 publications

References 24 publications

A model of the molten globule state from molecular dynamics simulations.

A model of the molten globule state from molecular dynamics simulations.

A peptide corresponding to the N‐terminal 13 residues of T4 lysozyme forms an α‐helix

Stabilizing the subtilisin BPN' pro‐domain by phage display selection: How restrictive is the amino acid code for maximum protein stability?

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