Impact of <i>Cis</i>‐proline analogs on peptide conformation

Che, Ye; Marshall, Garland R.

doi:10.1002/bip.20431

Cited by 60 publications

(46 citation statements)

References 92 publications

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“…The stability of β-turns within protein structures can be increased by incorporating Dproline (19) and certain β-amino acids (20) that bias the conformations occupied by the unfolded polypeptide. Likewise, proteins containing a cis-peptide bond can be stabilized by constrained amides or isosteres (21)(22)(23). The utility of these substitutions is limited, however, by the difficulty of modifying the backbone of proteins as well as concomitant effects on structure and function (24)(25)(26)(27).…”

mentioning

confidence: 99%

Stereoelectronic and steric effects in side chains preorganize a protein main chain

Shoulders¹,

Satyshur²,

Forest³

et al. 2009

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

153

142

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Preorganization is shown to endow a protein with extraordinary conformational stability. This preorganization is achieved by installing side-chain substituents that impose stereoelectronic and steric effects that restrict main-chain torsion angles. Replacing proline residues in ðProProGlyÞ 7 collagen strands with 4-fluoroproline and 4-methylproline leads to the most stable known triple helices, having T m values that are increased by >50°C. Differential scanning calorimetry data indicate an entropic basis to the hyperstability, as expected from an origin in preorganization. Structural data at a resolution of 1.21 Å reveal a prototypical triple helix with insignificant deviations to its main chain, even though 2∕3 of the residues are nonnatural. Thus, preorganization of a main chain by subtle changes to side chains can confer extraordinary conformational stability upon a protein without perturbing its structure.collagen triple helix | nonnatural amino acid | preorganization | protein stability | x-ray crystallography T he three-dimensional structures of proteins are stable because of a delicate balance of forces (1). Increases in the conformational stability of a protein-desirable in many contexts (2)-can be realized by enhancing the hydrophobic effect (3, 4), introducing or shielding a hydrogen bond (5) or electrostatic interaction (6-8), or introducing a disulfide crosslink (9-11) or metal ion-binding site (12). These strategies are often frustrated by enthalpy-entropy compensation, whereby enthalpic stabilization is offset entirely by entropic destabilization, or vice versa (13). Moreover, the strategies often lack subtlety and can lead to a misshapen and hence dysfunctional protein.As elaborated by Cram (14), the principle of preorganization states that, "the more highly hosts and guests are organized for binding and low solvation prior to their complexation, the more stable will be their complexes." In a typical manifestation, a conformational constraint is imposed upon a receptor or ligand during its chemical synthesis. This principle can also yield biopolymers with increased conformational stability (Fig. 1). For example, the stability of a DNA duplex correlates with the helicity of its single strands (15, 16), and a bicyclic ("locked") derivative of ribose enhances that stability (17, 18). The stability of β-turns within protein structures can be increased by incorporating Dproline (19) and certain β-amino acids (20) that bias the conformations occupied by the unfolded polypeptide. Likewise, proteins containing a cis-peptide bond can be stabilized by constrained amides or isosteres (21-23). The utility of these substitutions is limited, however, by the difficulty of modifying the backbone of proteins as well as concomitant effects on structure and function (24-27).We suspected that alterations to proline residues could provide a particularly incisive means to preorganize the conformation of a polypeptide chain. In classic work, Matthews and coworkers substituted proline, which is the least flexible residu...

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mentioning

confidence: 99%

Stereoelectronic and steric effects in side chains preorganize a protein main chain

Shoulders¹,

Satyshur²,

Forest³

et al. 2009

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

153

142

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“…Interestingly, structurally altered Pro derivatives, e.g. the cis-Pro analog thiaproline (63), accelerate the Pmn reaction of OmpA-(1-153)-Pro-tRNA Pro (22), suggesting that the unusually low reactivity of Pro may be due to its restricted conformational flexibility.…”

mentioning

confidence: 99%

Modulation of the Rate of Peptidyl Transfer on the Ribosome by the Nature of Substrates

Wohlgemuth

Brenner

Beringer

et al. 2008

Journal of Biological Chemistry

157

194

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The ribosome catalyzes peptide bond formation between peptidyl-tRNA in the P site and aminoacyl-tRNA in the A site. Here, we show that the nature of the C-terminal amino acid residue in the P-site peptidyl-tRNA strongly affects the rate of peptidyl transfer. Depending on the C-terminal amino acid of the peptidyl-tRNA, the rate of reaction with the small A-site substrate puromycin varied between 100 and 0.14 s ؊1 , regardless of the tRNA identity. The reactivity decreased in the order Lys ‫؍‬ Arg > Ala > Ser > Phe ‫؍‬ Val > Asp Ͼ Ͼ Pro, with Pro being by far the slowest. However, when Phe-tRNA Phe was used as A-site substrate, the rate of peptide bond formation with any peptidyl-tRNA was ϳ7 s ؊1 , which corresponds to the rate of binding of Phe-tRNA Phe to the A site (accommodation). Because accommodation is rate-limiting for peptide bond formation, the reaction rate is uniform for all peptidyltRNAs, regardless of the variations of the intrinsic chemical reactivities. On the other hand, the 50-fold increase in the reaction rate for peptidyl-tRNA ending with Pro suggests that full-length aminoacyl-tRNA in the A site greatly accelerates peptide bond formation.The enzymatic activity of the ribosome is to catalyze peptide bond formation. During the peptidyl transfer reaction, the ␣-amino group of aminoacyl-tRNA bound to the A site of the ribosome attacks the ester bond of peptidyl-tRNA in the P site, which results in peptidyl-tRNA extended by one amino acid in the A site and deacylated tRNA in the P site. The tRNA substrates are aligned in the active center of the ribosome by interactions of their CCA ends with 23 S rRNA bases (1-3). The ribosome lowers the activation entropy of the reaction (4, 5) by orienting the reacting groups precisely relative to each other (2, 3), providing an electrostatic environment that reduces the free energy of forming the transition state, shielding the reaction against bulk water (6, 7), or a combination of these effects (8).The peptidyl transfer reaction is modulated by conformational changes at the active site (3, 8 -10) as well as by the nature of the substrates. Rapid peptide bond formation requires fulllength tRNA in both A and P sites, and the reaction rate is influenced by the length of the tRNA fragments when model substrates are used (8, 10 -14). The reaction rate is also influenced by the nature of the amino acid side chain of the A-site substrate (13,(15)(16)(17), but is independent of the nucleophilicity of the attacking amino group in model substrates (18). Moreover, the length of the peptidyl chain and the nature of the C-terminal amino acid of the peptidyl-tRNA in the P site seem to have an effect (10,12,13,19). Early studies with 50 S ribosomal subunits indicated that efficient peptidyl transfer was observed with 3Ј-terminal RNase T1 fragments of N-acetylArg-tRNA Arg and fMet-tRNA fMet as model P-site substrates and an analog of aminoacyl-tRNA, puromycin (Pmn 4 ; O-methyltyrosine linked to N 6 -dimethyladenosine via an amide bond), as A-site substrate (20). In contrast,...

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“…19 The discussion centered on generating a construct similar to RNase S, but which explored the role of the C-terminal segment of RNase contributing His119 to the catalytic site. Ultimately, the semisynthetic ribonuclease, RNase-(1-118)- (111)(112)(113)(114)(115)(116)(117)(118)(119)(120)(121)(122)(123)(124), was evolved to study the role of the C-terminal segment in recognition and catalysis. [45][46][47][48] A three-component system, combining equimolar amounts of RNase fragments 1-20, 21-118, and 111-124, retained 30% of the enzymatic activity of intact RNase.…”

Section: Complex Of Rnase With C-terminal Segmentsmentioning

confidence: 99%

“…The question of interest, however, is in the actual entropic change introduced by either preorganization of the ligand and/or binding to another molecule as determined experimentally or by computation. In a similar study, Arnold et al 117 introduced 5,5-dimethyl-L-proline (dmP) for Pro114 to stabilize the cis-amide bond 118 between residues 113 and 114 by EPL (Figure 13). The catalytic activity k cat /K m was indistinguishable from that of the native enzyme.…”

Section: Torsional Entropymentioning

confidence: 99%

“…Other proline analogs impact cis-trans amide isomerization and, therefore, type-VI reverse-turn formation. 118,122 An excellent geometrical mimic of the cis-amide bond, the 1,5-disubstituted tetrazole ring, has been used by Zabrocki and coworkers [123][124][125] to probe type-VI reverse-turn recognition. Incorporation of a series of such …”

Section: Torsional Entropymentioning

confidence: 99%

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Back to the future: Ribonuclease A

2008

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Pancreatic ribonuclease A (EC 3.1.27.5, RNase) is, perhaps, the best‐studied enzyme of the 20th century. It was isolated by René Dubos, crystallized by Moses Kunitz, sequenced by Stanford Moore and William Stein, and synthesized in the laboratory of Bruce Merrifield, all at the Rockefeller Institute/University. It has proven to be an excellent model system for many different types of experiments, both as an enzyme and as a well‐characterized protein for biophysical studies. Of major significance was the demonstration by Chris Anfinsen at NIH that the primary sequence of RNase encoded the three‐dimensional structure of the enzyme. Many other prominent protein chemists/enzymologists have utilized RNase as a dominant theme in their research. In this review, the history of RNase and its offspring, RNase S (S‐protein/S‐peptide), will be considered, especially the work in the Merrifield group, as a preface to preliminary data and proposed experiments addressing topics of current interest. These include entropy–enthalpy compensation, entropy of ligand binding, the impact of protein modification on thermal stability, and the role of protein dynamics in enzyme action. In continuing to use RNase as a prototypical enzyme, we stand on the shoulders of the giants of protein chemistry to survey the future. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 259–277, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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Impact of Cis‐proline analogs on peptide conformation

Cited by 60 publications

References 92 publications

Stereoelectronic and steric effects in side chains preorganize a protein main chain

Stereoelectronic and steric effects in side chains preorganize a protein main chain

Modulation of the Rate of Peptidyl Transfer on the Ribosome by the Nature of Substrates

Back to the future: Ribonuclease A

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