Long-Range Interactions Within a Nonnative Protein

Klein‐Seetharaman, Judith; Oikawa, Maki; Grimshaw, Shaun B.; Wirmer, Julia; Duchardt, Elke; Ueda, Tadashi; Imoto, Taiji; Smith, Lorna J.; Dobson, Christopher M.; Schwalbe, Harald

doi:10.1126/science.1067680

Cited by 610 publications

(755 citation statements)

References 36 publications

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“…Baldwin and coworkers 64,65 described ion pairs and helix dipole interactions in the S-peptide of RNase, which were not expected to be maintained in the native state of RNase. Dobson, Schwalbe and coworkers 66 observed long-range nonnative interactions in lysozyme unfolding in 8 M urea, which were related to a non-native Arg-TrpArg sandwich structure, as found by Zhou and coworkers 47 with microseconds of MD simulations.…”

Section: Resultsmentioning

confidence: 58%

β‐strand interactions at the domain interface critical for the stability of human lens γD‐crystallin

2009

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Human age-onset cataracts are believed to be caused by the aggregation of partially unfolded or covalently damaged lens crystallin proteins; however, the exact molecular mechanism remains largely unknown. We have used microseconds of molecular dynamics simulations with explicit solvent to investigate the unfolding process of human lens cD-crystallin protein and its isolated domains. A partially unfolded folding intermediate of cD-crystallin is detected in simulations with its C-terminal domain (C-td) folded and N-terminal domain (N-td) unstructured, in excellent agreement with biochemical experiments. Our simulations strongly indicate that the stability and the folding mechanism of the N-td are regulated by the interdomain interactions, consistent with experimental observations. A hydrophobic folding core was identified within the C-td that is comprised of a and b strands from the Greek key motif 4, the one near the domain interface. Detailed analyses reveal a surprising non-native surface salt-bridge between Glu135 and Arg142 located at the end of the ab folded hairpin turn playing a critical role in stabilizing the folding core. On the other hand, an in silico single E135A substitution that disrupts this non-native Glu135-Arg142 salt-bridge causes significant destabilization to the folding core of the isolated C-td, which, in turn, induces unfolding of the N-td interface. These findings indicate that certain highly conserved charged residues, that is, Glu135 and Arg142, of cD-crystallin are crucial for stabilizing its hydrophobic domain interface in native conformation, and disruption of charges on the cD-crystallin surface might lead to unfolding and subsequent aggregation.

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Section: Resultsmentioning

confidence: 58%

β‐strand interactions at the domain interface critical for the stability of human lens γD‐crystallin

2009

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“…On the other hand, it has been confirmed in recent years that even proteins under highly denaturing conditions with reduced disulfide bonds keep a non-random structural preference for the formation of secondary and tertiary structures (49). Also, hydrophobic interactions persist under strongly denaturing conditions (50), and compact molten globule structures can be present in the absence of disulfides (51). A similar phenomenon is observed as the concentration of denaturant is increased in the oxidative folding of AAI.…”

Section: Discussionmentioning

confidence: 74%

Oxidative Folding of Amaranthus α-Amylase Inhibitor

Cemazar

Zahariev

Pongor

et al. 2004

Journal of Biological Chemistry

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Oxidative folding is the fusion of native disulfide bond formation with conformational folding. This complex process is guided by two types of interactions: first, covalent interactions between cysteine residues, which transform into native disulfide bridges, and second, non-covalent interactions giving rise to secondary and tertiary protein structure. The aim of this work is to understand both types of interactions in the oxidative folding of Amaranthus ␣-amylase inhibitor (AAI) by providing information both at the level of individual disulfide species and at the level of amino acid residue conformation. The cystine-knot disulfides of AAI protein are stabilized in an interdependent manner, and the oxidative folding is characterized by a high heterogeneity of one-, two-, and three-disulfide intermediates. The formation of the most abundant species, the main folding intermediate, is favored over other species even in the absence of non-covalent sequential preferences. Time-resolved NMR and photochemically induced dynamic nuclear polarization spectroscopies were used to follow the oxidative folding at the level of amino acid residue conformation. Because this is the first time that a complete oxidative folding process has been monitored with these two techniques, their results were compared with those obtained at the level of an individual disulfide species. The techniques proved to be valuable for the study of conformational developments and aromatic accessibility changes along oxidative folding pathways. A detailed picture of the oxidative folding of AAI provides a model study that combines different biochemical and biophysical techniques for a fuller understanding of a complex process.Cracking the code for folding a string of amino acid residues into a biologically active three-dimensional structure is still a major challenge of both chemical and biological interest. The information that determines the folding of a protein is contained solely in its amino acid residues (1). In proteins that contain cysteine residues the formation of disulfide bonds is an additional degree of freedom in the folding process. In these proteins a fusion between the recovery of the native tertiary structure (conformational folding) and the regeneration of native disulfide bonds is coupled in the oxidative folding process (2).Disulfide formation is important for in vivo folding of a considerable number of eukaryotic proteins and is a key posttranslational modification for the stabilization of folded structures. The study of oxidative folding in vitro can provide a detailed structural description in terms of the disulfide intermediate species present along the pathway of the complex folding process. A thorough account of an oxidative folding reaction should combine a study of the formation of covalently stable intermediate species and their disulfide bond connectivity and an analysis of the conformational assembly of these species. An established method for elucidation of the exact role that disulfides play in the process of achieving...

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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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Long-Range Interactions Within a Nonnative Protein

Cited by 610 publications

References 36 publications

β‐strand interactions at the domain interface critical for the stability of human lens γD‐crystallin

β‐strand interactions at the domain interface critical for the stability of human lens γD‐crystallin

Oxidative Folding of Amaranthus α-Amylase Inhibitor

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

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