Mutagenic and Thermodynamic Analyses of Residual Structure in the α Subunit of Tryptophan Synthase

Saab‐Rincón, Gloria; Pj, Gualfetti; Cr, Matthews

doi:10.1021/bi951726o

Cited by 63 publications

(65 citation statements)

References 37 publications

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“…99 The persistence of hydrophobic clusters at high denaturant concentration has been well documented. 3,111,112 Figure 15. PRE rates as experimentally measured in the sample of R74C-MTSL ubiquitin in 8M urea (pH 2, protein concentration 0.35 mM, reference compound N-acetylglycine, temperature 5 C, proton frequency 600 MHz; see Supporting Information for additional information).…”

Section: Resultsmentioning

confidence: 99%

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

et al. 2009

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Site-directed spin labeling in combination with paramagnetic relaxation enhancement (PRE) measurements is one of the most promising techniques for studying unfolded proteins. Since the pioneering work of Gillespie and Shortle (J Mol Biol 1997;268:158), PRE data from unfolded proteins have been interpreted using the theory that was originally developed for rotational spin relaxation. At the same time, it can be readily recognized that the relative motion of the paramagnetic tag attached to the peptide chain and the reporter spin such as 1 H N is best described as a translation. With this notion in mind, we developed a number of models for the PRE effect in unfolded proteins: (i) mutual diffusion of the two tethered spheres, (ii) mutual diffusion of the two tethered spheres subject to a harmonic potential, (iii) mutual diffusion of the two tethered spheres subject to a simulated mean-force potential (Smoluchowski equation); (iv) explicit-atom molecular dynamics simulation. The new models were used to predict the dependences of the PRE rates on the 1 H N residue number and static magnetic field strength; the results are appreciably different from the Gillespie-Shortle model. At the same time, the Gillespie-Shortle approach is expected to be generally adequate if the goal is to reconstruct the distance distributions between 1 H N spins and the paramagnetic center (provided that the characteristic correlation time is known with a reasonable accuracy). The theory has been tested by measuring the PRE rates in three spin-labeled mutants of the drkN SH3 domain in 2M guanidinium chloride. Two modifications introduced into the measurement scheme-using a reference compound to calibrate the signals from the two samples (oxidized and reduced) and using peak volumes instead of intensities to determine the PRE rates-lead to a substantial improvement in the quality of data. The PRE data from the denatured drkN SH3 are mostly consistent with the model of moderately expanded random-coil protein, although part of the data point toward a more compact structure (local hydrophobic cluster). At the same time, the radius of gyration reported by Choy et al. (J Mol Biol 2002;316:101) suggests that the protein is highly expanded. This seemingly contradictory evidence can be reconciled if one assumes that denatured drkN SH3 forms a conformational ensemble that is dominated by extended conformations, yet also contains compact (collapsed) species. Such behavior is apparently more complex than predicted by the model of a random-coil protein in good solvent/poor solvent.Additional Supporting Information may be found in the online version of this article.Abbreviations: drkN SH3, N-terminal SH3 domain of the Drosophila adapter protein drk; EDTA, ethylenediaminetetraacetic acid; ESR, electron spin resonance; FRET, fluorescence resonance energy transfer; MTSL, methanethiosulfonate spin label; NOE, nuclear Overhauser effect; NAG, N-acetyl-glycine.

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Section: Resultsmentioning

confidence: 99%

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

et al. 2009

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“…Several recent studies have argued that unfolded states can have considerable residual structure even at elevated denaturant concentrations [18,19,20,21,22]. To a first approximation, unfolded states can be modeled as random coils.…”

Section: High Degree Of Packing In Proteins Correlates With Substantimentioning

confidence: 99%

Asymmetry in the Shapes of Folded and Denatured States of Proteins

2004

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The asymmetry in the shapes of folded and unfolded states are probed using two parameters, ∆ (a measure of the sphericity) and S that describes the shape (S > 0 corresponds to prolate and S < 0 represents oblate). For the folded states, whose interiors are densely packed, the radii of gyration (R g ), ∆, and S are calculated using the coordinates of the experimentally determined structures. Although R g scales as N 1/3 , as expected for maximally compact structures, the distributions of ∆ and S show that there is considerable asymmetry in the shapes of folded structures. The degree of asymmetry is greater for proteins that form oligomers. Analysis of the two-and three-body contacts in the native structures shows that the presence of near equal number of contacts between backbone and side-chains and between side-chains gives rise to dense packing. We suggest that proteins with relatively large values of ∆ and S can tolerate volume mutations without greatly affecting the network of contacts or their stability. Mutagenesis data on T4 lysozyme and λ-repressor support this conclusion. To probe shape characteristics of denatured states we have developed a C α − C β model of a WW-like domain.The shape parameters, which are calculated using Langevin simulations, change dramatically in the course of coil to globule transition. Comparison of the values of ∆ and S between the globular state and the folded state of WW domain shows that both energetic (especially dispersion in the hydrophobic interactions) and steric effects are important in determining packing in proteins.

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“…Thus, the three histidines might form a hydrophobic cluster with other hydrophobic residues (Tyr10, Val12, Leu17) at neutral pH. [21] However, significant chemical-shift changes were observed at lower pH values (7.0-5.8) not only for the three histidines but also for the basic residues Arg5 and Lys16 (Figure 2 A), presumably due to the protonation of the histidines. This suggests that cation-p interactions [22] between the side chains in the N terminus might also contribute to the stabilization of this cluster.…”

mentioning

confidence: 96%

A Weakly Clustered N terminus Inhibits Aβ(1–40) Amyloidogenesis

Lim

2006

ChemBioChem

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Mutagenic and Thermodynamic Analyses of Residual Structure in the α Subunit of Tryptophan Synthase

Cited by 63 publications

References 37 publications

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

Asymmetry in the Shapes of Folded and Denatured States of Proteins

A Weakly Clustered N terminus Inhibits Aβ(1–40) Amyloidogenesis

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