Local fluctuations vs. global unfolding of proteins investigated by limited proteolysis

Arnold, Ulrich; Köditz, Jens; Markert, Yvonne; Ulbrich‐Hofmann, Renate

doi:10.1080/10242420500183287

Cited by 13 publications

(12 citation statements)

References 38 publications

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“…As for the unfolding process, the section from the C-terminal end of helix II (Lys31) through the first β-sheet strand (Phe46) became susceptible first to proteolytic attack by thermolysin and trypsin under denaturing conditions [25] and several residues in this region showed a faster H-D exchange than that of global unfolding [26,27]. The designation of this region as unfolding region of RNase A was confirmed by both computer simulations and studies using either the glycosylated variant of RNase A, RNase B, or genetically engineered RNase A variants [28][29][30].…”

Section: Accepted M Manuscriptmentioning

confidence: 81%

The effect of additional disulfide bonds on the stability and folding of ribonuclease A

Pecher¹,

Arnold²

2009

Biophysical Chemistry

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International audienceThe significant contribution of disulfide bonds to the conformational stability of proteins is generally considered to result from an entropic destabilization of the unfolded state causing a faster escape of the molecules to the native state. However, the introduction of extra disulfide bonds into proteins as a general approach to protein stabilization yields rather inconsistent results. By modeling studies, we selected positions to introduce additional disulfide bonds into ribonuclease A at regions that had proven to be crucial for the initiation of the folding or unfolding process, respectively. However, only two out of the six variants proved to be more stable than unmodified ribonuclease A. The comparison of the thermodynamic and kinetic data disclosed a more pronounced effect on the unfolding reaction for all variants regardless of the position of the extra disulfide bond. Native-state proteolysis indicated a perturbation of the native state of the destabilized variants that obviously counterbalances the stability gain by the extra disulfide bond

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Section: Accepted M Manuscriptmentioning

confidence: 81%

The effect of additional disulfide bonds on the stability and folding of ribonuclease A

Pecher¹,

Arnold²

2009

Biophysical Chemistry

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“…The use of proteolytic digestion to probe dynamic behavior of proteins is well established and provides an alternative approach to spectroscopic means for obtaining thermodynamic information (45,50,61,62). However, the advantages it provides (simplicity despite structural size and complexity; amenability to varying buffer, pH, and temperature conditions; and sensitivity to large-scale motion) have not been fully explored.…”

Section: Resultsmentioning

confidence: 99%

“…These concerns make it vital that measurements of dynamic motion based on the disappearance of starting material be the result of a single cleavage pathway. Previously this requirement has been met by limiting the application of kinetic hydrolysis to small proteins that are well folded (61,62,65). Supramolecular complexes can be amenable to the same analyses, provided suitable precautions are met.…”

Section: Discussionmentioning

confidence: 99%

“…The application of kinetic hydrolysis can only resolve the rate of transition across an arbitrary boundary between protease-protected and accessible conformations. A two-state approximation has been a sufficient and successful model for kinetic hydrolysis studies of other proteins (60,62,65,70,71). Our results reaffirm applicability of a two-state closed/open system to HBV Cp149 dimer and capsid.…”

Section: Discussionmentioning

confidence: 99%

“…Upon local or global unfolding (deprotection) the cleavage site adopts the 'open' state and is exposed to hydrolytic attack. This two-state model has had wide acceptance for a variety of protein systems (50,(60)(61)(62), and the observed kinetic trends for Cp149 validate the two-state model for this application (or details, see Discussion).…”

Section: Kinetic Modelmentioning

confidence: 99%

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Conformational Equilibria and Rates of Localized Motion within Hepatitis B Virus Capsids

Hilmer

Zlotnick

Bothner

2008

Journal of Molecular Biology

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Functional analysis of Hepatitis B virus (HBV) core particles has associated a number of biological roles with the C-terminus of the capsid protein. One set of functions require the C-terminus to be on the exterior of the capsid, while others place this domain on the interior. According to the crystal structure of the capsid, this segment is strictly internal to the capsid shell and buried at a proteinprotein interface. Using kinetic hydrolysis, a form of protease digestion assayed by SDS-PAGE and mass spectrometry, the structurally and biologically important C-terminal region of HBV capsid protein assembly domain (Cp149, residues 1-149) has been shown to be dynamic in both dimer and capsid forms. HBV is an enveloped virus with a T=4 icosahedral core that is composed of 120 copies of a homodimer capsid protein. Free dimer and assembled capsid forms of the protein are readily hydrolyzed by trypsin and thermolysin, around residues 127-128, indicating that this region is dynamic and exposed to the capsid surface. The measured conformational equilibria have an opposite temperature dependence between free dimer and assembled capsid. This work helps to explain the previously described allosteric regulation of assembly and functional properties of a buried domain. These observations make a critical connection between structure, dynamics, and function: made possible by the first quantitative measurements of conformational equilibria and rates of conversion between protein conformers for a megadalton complex.

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The stability of engineered thermostable neutral proteases from Bacillus stearothermophilus in organic solvents and detergents

Mansfeld

Ulbrich‐Hofmann

2007

Biotech & Bioengineering

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Engineered extremely thermostable variants of the thermolysin-like protease from Bacillus stearothermophilus possessing an introduced disulfide bond G8C/N60C (double mutant, DM) and six additional amino acid substitutions in the exposed loop region 56-69 (Boilysin, BLN) have been probed with respect to stability toward water-miscible organic solvents and detergents. The solvent concentrations where 50% of enzyme activity were irreversibly lost (C(50)) decreased in the order methanol > 2-propanol > dimethylsulfoxide > dioxane > acetonitrile > dimethylformamide > acetone. The C(50) values were remarkably higher for the thermostable variants than for the wild-type enzymes. Therefore, the stabilization of this loop region also protects the molecule from irreversible inactivation by solvents, and inactivation seems to follow principally the same mechanism as thermal inactivation. However, in contrast to thermal inactivation where the corresponding T(50) values of DM and BLN differed by 10 K, the differences of the C(50) values of DM and BLN were not significant. Detergents had great effects on proteolytic activities which were dependent on the individual detergent and its concentration, but mostly without significant differences between the enzyme variants. These effects were inactivating (SDS, sulfobetaine) or strongly activating (CTAB, CHAPS). Triton X-100 and Tween 20 were activating or inactivating at low and high concentrations, respectively. In all detergents, stabilities of the enzymes were strongly decreased. However, the more thermostable variants were affected by the detergents to the same extent as the wild-type enzymes suggesting that the mechanism of detergent inactivation is different from that of thermal inactivation.

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Local fluctuations vs. global unfolding of proteins investigated by limited proteolysis

Cited by 13 publications

References 38 publications

The effect of additional disulfide bonds on the stability and folding of ribonuclease A

The effect of additional disulfide bonds on the stability and folding of ribonuclease A

Conformational Equilibria and Rates of Localized Motion within Hepatitis B Virus Capsids

The stability of engineered thermostable neutral proteases from Bacillus stearothermophilus in organic solvents and detergents

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