Accretion of Structure in Staphylococcal Nuclease: An15N NMR Relaxation Study

Alexandrescu, Andrei T.; Jahnke, Wolfgang; Wiltscheck, Ronald; Blommers, Marcel J. J.

doi:10.1006/jmbi.1996.0422

Cited by 46 publications

(65 citation statements)

References 41 publications

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“…The change in the fraction of a-helix between 10°C and 35°C corresponds to a DDG of only about À0.24 kcal/mol and a van't Hoff DH of about +1.1 kcal/mol. Depending on the kinetics of interconversion between conformational states even a small population of a-helix could give rise to large exchange broadening contributions (Alexandrescu et al 1996;Kempf and Loria 2004). To test for dynamics in the micro-to millisecond exchange regime we measured 15 N R2 ex terms for monomeric aS at pH 5.9 at both 10°C and 35°C.…”

Section: Secondary Chemical Shifts Indicate Only Small Amounts Of Resmentioning

confidence: 99%

“…To cause broadening of peaks beyond detection the R2 ex terms would probably have to be one or two orders of magnitude larger than what we observed. Typical of highly unfolded proteins aS appears to be dynamically flexible on nanoand sub-nanosecond timescales but lacks the slower microto millisecond motions that often accompany the formation of nascent folded structure (Alexandrescu et al 1996).…”

Section: Secondary Chemical Shifts Indicate Only Small Amounts Of Resmentioning

confidence: 99%

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Hydrogen exchange of monomeric α‐synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli

et al. 2008

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Amide proton NMR signals from the N-terminal domain of monomeric a-synuclein (aS) are lost when the sample temperature is raised from 10°C to 35°C at pH 7.4. Although the temperature-induced effects have been attributed to conformational exchange caused by an increase in a-helix structure, we show that the loss of signals is due to fast amide proton exchange. At low ionic strength, hydrogen exchange rates are faster for the N-terminal segment of aS than for the acidic C-terminal domain. When the salt concentration is raised to 300 mM, exchange rates increase throughout the protein and become similar for the N-and C-terminal domains. This indicates that the enhanced protection of amide protons from the C-terminal domain at low salt is electrostatic in nature. Ca chemical shift data point to <10% residual a-helix structure at 10°C and 35°C. Conformational exchange contributions to R2 are negligible at both temperatures. In contrast to the situation in vitro, the majority of amide protons are observed at 37°C in 1 H-15 N HSQC spectra of aS encapsulated within living Escherichia coli cells. Our finding that temperature effects on aS NMR spectra can be explained by hydrogen exchange obviates the need to invoke special cellular factors. The retention of signals is likely due to slowed hydrogen exchange caused by the lowered intracellular pH of high-density E. coli cultures. Taken together, our results emphasize that aS remains predominantly unfolded at physiological temperature and pH-an important conclusion for mechanistic models of the association of aS with membranes and fibrils. Abbreviations: aS, a-synuclein; ct, constant time; CLEANEX, clean chemical exchange; DSS, 2,2-Dimethyl-2-silapentane-5-sulfonic acid; HSQC, heteronuclear single quantum coherence spectroscopy; GM1, monosialotetrahexosylganglioside; R2, transverse relaxation rate (1/ T2); R2 ex , chemical exchange contribution to R2; SDS, sodium dodecyl sulfate.Article and publication are at http://www.proteinscience.org/cgi

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Section: Secondary Chemical Shifts Indicate Only Small Amounts Of Resmentioning

confidence: 99%

Section: Secondary Chemical Shifts Indicate Only Small Amounts Of Resmentioning

confidence: 99%

Hydrogen exchange of monomeric α‐synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli

et al. 2008

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“…Examples include, in particular RMSD values of NMR structures (Redfield et al, 1992;Buck et al, 1995;Alexandrescu et al, 1996;Fushman et al, 1997); and other structural parameters such as 35 coupling constants, and chemical shift deviations from "coil" values (Buck et al, 1995;Alexandrescu et al, 1996). The question of whether a quantitative correspondence exists has not yet been examined in detail; nevertheless, a qualitative relationship is suggested.…”

Section: A Possible Advantage Of the Mf Approach Is That In Many Casesmentioning

confidence: 99%

“…The samples contained no added buffers or salts. Backbone "N R1, R2, and NOE NMR relaxation data were obtained using previously published experiments (Farrow et al, 1994;Alexandrescu et al, 1996). For the free S-peptide T1 and T2 relaxation parameters were measured from 8 data sets: T1 relaxation periods were 0.1, 0.2,0.4,0.5,0.6,0.7,0.8, and 1.0 s; T2 relaxation periods were 16, 32, 6 4 , 128,200,256,300, and 400 ms.…”

Section: Methodsmentioning

confidence: 99%

“…At the present time NMR relaxation studies of highly dynamic polypeptides are few in number (Torchia et al, 1975;vanMierlo et al, 1993;Logan et al, 1994;Frank et al, 1995;Alexandrescu et al, 1996;Buck et al, 1996;Farrow et al, 1997;Kemple et al, 1997;Schwalbe et al, 1997). One of the most interesting trends emerging from these studies is the extremely diverse behavior of proteins commonly lumped together under the designation "unfolded."…”

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¹⁵N backbone dynamics of the S‐peptide from ribonuclease A in its free and S‐protein bound forms: Toward a site‐specific analysis of entropy changes upon folding

et al. 1998

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Backbone 15N relaxation parameters (Rl, R2, 1H‐15N NOE) have been measured for a 22‐residue recombinant variant of the S‐peptide in its free and S‐protein bound forms. NMR relaxation data were analyzed using the “model‐free” approach (Lipari & Szabo, 1982). Order parameters obtained from “model‐free” simulations were used to calculate 1H‐15N bond vector entropies using a recently described method (Yang & Kay, 1996), in which the form of the probability density function for bond vector fluctuations is derived from a diffusion‐in‐a‐cone motional model. The average change in 1H‐15N bond vector entropies for residues T3‐S15, which become ordered upon binding of the S‐peptide to the S‐protein, is ‐12.6 ± 1.4 J/mol.residue.K. 15N relaxation data suggest a gradient of decreasing entropy values moving from the termini toward the center of the free peptide. The difference between the entropies of the terminal and central residues is about ‐ 12 J/mol.residue.K, a value comparable to that of the average entropy change per residue upon complex formation. Similar entropy gradients are evident in NMR relaxation studies of other denatured proteins. Taken together, these observations suggest denatured proteins may contain entropic contributions from non‐local interactions. Consequently, calculations that model the entropy of a residue in a denatured protein as that of a residue in a di ‐ or tri‐peptide, might over‐estimate the magnitude of entropy changes upon folding.

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Correlation between changes in nuclear magnetic resonance order parameters and conformational entropy: Molecular dynamics simulations of native and denatured staphylococcal nuclease

2000

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Recent work has suggested that changes in NMR order parameters may quantitatively reflect changes in the conformational entropy of a protein ensemble. The extent of the mathematical relationship between local entropy changes as seen by NMR order parameters and the full protein entropy change is a complex issue. As a step towards a fuller understanding of this problem, molecular dynamics calculations of both native and denatured staphylococcal nuclease were performed. The N-H bond vector motion, in both explicit and implicit solvent, was analyzed to estimate local and global entropy changes. The calculated N-H bond vector order parameters from simulation agreed on average with experimental values for both native and denatured structures. However, the inverted-U profile of order parameters versus residue number observed experimentally for denatured nuclease was only partially reproduced by simulation of compact denatured structures. Comparisons made across the full set of simulations revealed a correlation between the N-H order parameter-based conformational entropy change and the total quasiharmonic-based conformational entropy change between the native and denatured structures. The calculations showed that about 25% of the total entropy change was reflected by changes in simulated S2 values. This result suggests that NMR-derived order parameters may be used to provide a reasonable estimate of the total conformational entropy change on protein folding.

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Accretion of Structure in Staphylococcal Nuclease: An15N NMR Relaxation Study

Cited by 46 publications

References 41 publications

Hydrogen exchange of monomeric α‐synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli

Hydrogen exchange of monomeric α‐synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli

¹⁵N backbone dynamics of the S‐peptide from ribonuclease A in its free and S‐protein bound forms: Toward a site‐specific analysis of entropy changes upon folding

Correlation between changes in nuclear magnetic resonance order parameters and conformational entropy: Molecular dynamics simulations of native and denatured staphylococcal nuclease

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Accretion of Structure in Staphylococcal Nuclease: An15N NMR Relaxation Study

Cited by 46 publications

References 41 publications

Hydrogen exchange of monomeric α‐synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli

Hydrogen exchange of monomeric α‐synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli

15N backbone dynamics of the S‐peptide from ribonuclease A in its free and S‐protein bound forms: Toward a site‐specific analysis of entropy changes upon folding

Correlation between changes in nuclear magnetic resonance order parameters and conformational entropy: Molecular dynamics simulations of native and denatured staphylococcal nuclease

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¹⁵N backbone dynamics of the S‐peptide from ribonuclease A in its free and S‐protein bound forms: Toward a site‐specific analysis of entropy changes upon folding