In a staphylococcal nuclease mutant the side-chain of a lysine replacing valine 66 is fully buried in the hydrophobic core

Stites, Wesley E.; Gittis, Apostolos G.; Lattman, Eaton E.; Shortle, David

doi:10.1016/0022-2836(91)80195-z

Cited by 163 publications

(190 citation statements)

References 22 publications

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“…Fourteen were eliminated because they were in unrefined structures (three), are charge compensated (eight), or lie mostly on the protein surface (three). The only examples of truly buried (no solvent accessibility) and uncompensated lysine side chains were found in glycogen phosphorylase and very recently in a staphylococcal nuclease mutant (Stites et al, 1991) and a T4 lysozyme mutant (Dao-pin et al, 1991). Thus, replacement of Ser214 in trypsin with lysine provides a rare example of a buried, formal charge.…”

Section: Resultsmentioning

confidence: 99%

Perturbing the polar environment of Asp102 in trypsin: consequences of replacing conserved Ser214

McGrath

Vásquez²,

Craik³

et al. 1992

Biochemistry

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Much of the catalytic power of trypsin is derived from the unusual buried, charged side chain of Aspl02. A polar cave provides the stabilization for maintaining the buried charge, and it features the conserved amino acid Ser214 adjacent to Aspl02. Ser214 has been replaced with Ala, Glu, and Lys in rat anionic trypsin, and the consequences of these changes have been determined. Three-dimensional structures of the Glu and Lys variant trypsins reveal that the new 214 side chains are buried. The 2.2-A crystal structure (R = 0.150) of trypsin S214K shows that Lys214 occupies the position held by Ser214 and a buried water molecule in the buried polar cave. Lys214-Nf is solvent inaccessible and is less than 5 A from the catalytic Aspl02. The side chain of Glu214 (2.8 A, R = 0.168) in trypsin S214E shows two conformations. In the major one, the Glu carboxylate in S214E forms a hydrogen bond with Aspl02. Analytical isoelectrofocusing results show that trypsin S214K has a significantly different isoelectric point than trypsin, corresponding to an additional positive charge. The kinetic parameter kat demonstrates that, compared to trypsin, S214K has 1% of the catalytic activity on a tripeptide amide substrate and S214E is 44% as active. Electrostatic potential calculations provide corroboration of the charge on Lys214 and are consistent with the kinetic results, suggesting that the presence of Lys214 has disturbed the electrostatic potential of Aspl02.Catalysis in the serine proteases is attributed to a triad of amino acids which orchestrate an attack by serine (Ser195 in trypsin) on the substrate carbonyl carbon.

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Section: Resultsmentioning

confidence: 99%

Perturbing the polar environment of Asp102 in trypsin: consequences of replacing conserved Ser214

McGrath

Vásquez²,

Craik³

et al. 1992

Biochemistry

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“…A survey of high-resolution structures indicates that accommodation of charge in a protein interior occurs relatively rarely and only when compensating hydrogen bondinglsalt bridging partners are available (Rashin & Honig, 1986). However, mutational analysis of T4 lysozyme (Daopin et al, 1991), staphylococcal nuclease (Stites et al, 1991), and E. coli thioredoxin (Hellinga et al, 1992) have identified buried positions that can tolerate potentially charged side chains. In the case of V66K nuclease, the pH dependence of stability suggests that the lysine residue is unprotonated in the folded state; i.e., there is a marked perturbation of the pK, of this residue in the folded state.…”

Section: Discussionmentioning

confidence: 99%

Interactions in nonnative and truncated forms of staphylococcal nuclease as indicated by mutational free energy changes

et al. 1995

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Several mixed disulfide variants of staphylococcal nuclease have been produced by disulfide bond formation between nuclease V23C and methane, ethane, l-propane, I-n-butane, and I-n-pentane thiols. Although CD spectroscopy shows that the native state is largely unperturbed, the stability toward urea-induced unfolding is highly dependent on the nature of the group at this position, with the methyl disulfide protein being the most stable. The variant produced by modification with iodoacetic acid, however, gives a CD spectrum indicative of an unfolded polypeptide. Thiol-disulfide exchange equilibrium constants between nuclease V23C and 2-hydroxyethyl disulfide have been measured as a function of urea concentration. Because thiol-disulfide exchange and unfolding are thermodynamically linked, the effects of a mutation (disulfide exchange) can be partitioned between various conformational states. In the case of unmodified V23C and the 2-hydroxyethyl protein mixed disulfide, significant effects in the nonnative states of nuclease are observed.Truncated forms of staphylococcal nuclease are thought to be partially folded and may be good models for early folding intermediates. We have characterized a truncated form of nuclease comprised of residues 1-135 with a V23C mutation after chemical modification of the cysteine residue. High-resolution size-exclusion chromatography indicates that modification brings about significant changes in the Stokes radius of the protein, and CD spectroscopy indicates considerable differences in the amount of secondary structure present. Measurement of the disulfide exchange equilibrium constant between this truncated protein and 2-hydroxyethyl disulfide indicate significant interactions between position 23 and the rest of the protein when the urea concentration is lower than 1.5 M. Comparison of disulfide exchange equilibrium constants for the truncated and nonnative full-length forms indicates that some of the interactions in unfolded nuclease are dependent upon residues far removed in sequence and not in contact in the native structure.Keywords: cysteine modification; thermodynamic cycle; truncated nuclease; unfolded stateThe stability of a protein is determined by the difference in free energy between the folded and unfolded forms. Although most researchers agree that mutations can cause large effects on the Reprint requests to: Richard

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“…However, given this narrow pK a window, there is no pH at which the ␣-amine can be selectively labeled with an amine-labeling reagent, because -amines, albeit less reactive, are typically much more abundant (10). The circumstances are further complicated by the fact that the pK a of the ammonium ion for the ␣-amine and the -amine can be affected by the side chain of the adjacent amino acids, hydrogen bonding, and other intramolecular interactions (11,12).…”

Section: A Chemoselective Strategy For Labeling and Enriching N-termimentioning

confidence: 99%

Global profiling of protease cleavage sites by chemoselective labeling of protein N-termini

Shin

Jaffrey

2009

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

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Proteolysis has major roles in diverse biologic processes and regulates the activity, localization, and intracellular levels of proteins. Linking signaling pathways and physiologic processes to specific proteolytic processing events is a major challenge in signal transduction research. Here, we describe N-CLAP (N-terminalomics by chemical labeling of the ␣-amine of proteins), a general approach for profiling protein N-termini and identifying protein cleavage sites during cellular signaling. In N-CLAP, simple and readily available reagents are used to selectively affinity label the ␣-amine that characterizes the protein N terminus over the more highly abundant -amine on lysine residues. Protein cleavage sites are deduced by identifying the corresponding N-CLAP peptides, which are derived from the N-termini of proteins, including the N-termini of the newly formed polypeptide products of proteolytic cleavage. Through selective affinity purification and tandem mass spectrometry analysis of 278 N-CLAP peptides, we characterized proteolytic cleavage events associated with methionine aminopeptidases and signal peptide peptidases, as well as proteins that are proteolytically cleaved after cisplatin-induced apoptosis. Many of the protein cleavage sites that are elicited during apoptotic signaling are consistent with caspase-dependent cleavage. These data demonstrate the utility of N-CLAP for proteomic profiling of protein cleavage sites that are generated during cellular signaling.caspase ͉ N-CLAP ͉ N-terminalomics ͉ tandem mass spectrometry

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In a staphylococcal nuclease mutant the side-chain of a lysine replacing valine 66 is fully buried in the hydrophobic core

Cited by 163 publications

References 22 publications

Perturbing the polar environment of Asp102 in trypsin: consequences of replacing conserved Ser214

Perturbing the polar environment of Asp102 in trypsin: consequences of replacing conserved Ser214

Interactions in nonnative and truncated forms of staphylococcal nuclease as indicated by mutational free energy changes

Global profiling of protease cleavage sites by chemoselective labeling of protein N-termini

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