Ribonucleases as Models for Understanding Protein Folding

Scheraga, Harold A.

doi:10.1007/978-3-642-21078-5_15

Cited by 3 publications

(8 citation statements)

References 119 publications

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“…The unfolding simulations of RNase A provide strong evidence that disulfide-bond formation/disruption is temperature-dependent except for the disulfide bond Cys65-Cys72. Experimental studies suggest that the entropy of formation for the Cys65-Cys72 disulfide bond is the lowest of the four disulfide bonds; , therefore, this bond is expected to be least dependent on temperature during the unfolding process, which is in agreement with our simulations (see section ). In general, performed simulations offer the view that formation/disruption of disulfide bonds influences the structure of RNase A during the unfolding progress and that distinct order of disulfide-bond breaking can be observed.…”

Section: Introductionsupporting

confidence: 91%

“…With an increase of temperature, the Cys85-Cys95 fragment becomes the primary source of fluctuations in the structure of RNase A, which affects the whole structure. Such a structure (Figure B) resembles the intermediate state found in the most common folding pathway of RNase A observed by Li et al , Using peptide mapping, another intermediate structure, lacking the Cys65-Cys72 disulfide bond, was observed by Rothwarf et al, which is also present in molecular dynamics simulations (Figure D). However, in general, the Cys65-Cys72 disulfide bond was found to be very stable in the simulations performed in this work and was also determined experimentally to be the most populated disulfide bond in one- and two-disulfide-bond intermediates. − The flexible region of residues 16–25, which is the loop between the first and second α-helix, is stabilized by the Cys26-Cys84 disulfide bond at all investigated temperatures, keeping fluctuations on the same level in the 278–308 K temperature range.…”

Section: Resultssupporting

confidence: 67%

“…The presence of multiple distinct unfolding pathways 15,48 and hence disulfide-bond disruption/formation is strictly connected to (un)folding pathways; 49 a distinctive order of disulfide-bond breaking was also observed experimentally and theoretically for RNase A and other proteins. 48,50,51 At all temperatures, the first disulfide bond to break is the Cys40-Cys95 bond (in at least 80% of the trajectories; see Table 1), forming the intermediate structures, also observed experimentally as the most populated intermediate structures, 15,28,52 especially in RNase A mutants, 53 which can form native-like structures. 54 Our kinetic analysis (for more details see section S1 'Kinetics and thermodynamics of disulfide bonds' in the Supporting Information) shows that the Cys40-Cys95 bond is, except for the simulation carried out at T = 278 K, at least an order of magnitude faster to break (Supporting Table S1 and Supporting Figure S4).…”

Section: Analysis Of Stability Of Rnasementioning

confidence: 59%

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Dynamics of Disulfide-Bond Disruption and Formation in the Thermal Unfolding of Ribonuclease A

Krupa

Sieradzan

Mozolewska

et al. 2017

J. Chem. Theory Comput.

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Ribonuclease A (RNase A) is the most studied member of ribonucleases − a group of enzymes responsible for catalyzing RNA degradation. RNase A contains four disulfide bonds, which were found to be necessary for the native structure of the protein to form. In this work the kinetics and thermodynamics of RNase unfolding were studied by means of a series of coarse-grained canonical molecular-dynamics simulations, run at various temperatures, and replica-exchange molecular dynamics simulations with the UNRES force field, which is capable of dynamic formation and breaking the disulfide bonds during the course of the simulations. It was found that the Cys40-Cys95 bond was the first to break, while the Cys26-Cys84 bond was the last to break, independent of temperature, in agreement with available experimental data. Except for the Cys40-Cys95 bond, all disulfide bonds were relatively stable during the simulations. The formation/disruption of disulfide bonds was found to be temperature dependent for three out of four disulfide bonds in RNase A, except for the most stable disulfide bond between Cys65 and Cys72. A stable intermediate without the Cys40-Cys95 disulfide bond, with structure similar to that of the most common folding intermediate observed experimentally, was found in simulated unfolding. In agreement with experiment, non-native disulfide bonds were also observed. By analyzing residue-position fluctuations, it was found that native disulfide bonds are located in the highly flexible regions of the protein, which is probably why their presence is necessary for the stability of RNase A.

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Section: Introductionsupporting

confidence: 91%

Section: Resultssupporting

confidence: 67%

Section: Analysis Of Stability Of Rnasementioning

confidence: 59%

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Dynamics of Disulfide-Bond Disruption and Formation in the Thermal Unfolding of Ribonuclease A

Krupa

Sieradzan

Mozolewska

et al. 2017

J. Chem. Theory Comput.

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“…These oxidative-folding experiments were carried out on RNase A, first, with oxidized-and-reduced glutathione (Scheraga, 2011b; Scheraga et al 1984, 1987) but, later, to simplify the number of folding intermediates, with oxidized-and-reduced dithiothreitol (Rothwarf et al 1998; Scheraga, 2011b). …”

Section: Folding Pathways Of Reduced Rnase Amentioning

confidence: 99%

“…The experimental work on identifying hydrogen bonds in native RNase A (Scheraga, 1967, 2011b) was accompanied by an experimental approach to identify the pathways from the unfolded form of this protein (with its disulfide bonds reduced to sulfhydryl groups) to the oxidatively folded protein to form its four native disulfide bonds. These oxidative-folding experiments were carried out on RNase A, first, with oxidized-and-reduced glutathione (Scheraga, 2011b; Scheraga et al 1984, 1987) but, later, to simplify the number of folding intermediates, with oxidized-and-reduced dithiothreitol (Rothwarf et al 1998; Scheraga, 2011b).…”

Section: Folding Pathways Of Reduced Rnase Amentioning

confidence: 99%

My 65 years in protein chemistry

Scheraga

2015

Quart. Rev. Biophys.

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This is a tour of a physical chemist through 65 years of protein chemistry from the time when emphasis was placed on the determination of the size and shape of the protein molecule as a colloidal particle, with an early breakthrough by James Sumner, followed by Linus Pauling and Fred Sanger, that a protein was a real molecule, albeit a macromolecule. It deals with the recognition of the nature and importance of hydrogen bonds and hydrophobic interactions in determining the structure, properties, and biological function of proteins until the present acquisition of an understanding of the structure, thermodynamics, and folding pathways from a linear array of amino acids to a biological entity. Along the way, with a combination of experiment and theoretical interpretation, a mechanism was elucidated for the thrombin-induced conversion of fibrinogen to a fibrin blood clot and for the oxidative-folding pathways of ribonuclease A. Before the atomic structure of a protein molecule was determined by x-ray diffraction or nuclear magnetic resonance spectroscopy, experimental studies of the fundamental interactions underlying protein structure led to several distance constraints which motivated the theoretical approach to determine protein structure, and culminated in the Empirical Conformational Energy Program for Peptides (ECEPP), an all-atom force field, with which the structures of fibrous collagen-like proteins and the 46-residue globular staphylococcal protein A were determined. To undertake the study of larger globular proteins, a physics-based coarse-grained UNited-RESidue (UNRES) force field was developed, and applied to the protein-folding problem in terms of structure, thermodynamics, dynamics, and folding pathways. Initially, single-chain and, ultimately, multiple-chain proteins were examined, and the methodology was extended to protein–protein interactions and to nucleic acids and to protein–nucleic acid interactions. The ultimate results led to an understanding of a variety of biological processes underlying natural and disease phenomena.

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Monitoring Guanidinium-Induced Structural Changes in Ribonuclease Proteins Using Raman Spectroscopy and 2D Correlation Analysis

2013

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Assessing the stability of proteins by comparing their unfolding profiles is a very important characterization and quality control step for any biopharmaceutical, and this is usually measured by fluorescence spectroscopy. In this paper we propose Raman spectroscopy as a rapid, noninvasive alternative analytical method and we shall show this has enhanced sensitivity and can therefore reveal very subtle protein conformational changes that are not observed with fluorescence measurements. Raman spectroscopy is a powerful nondestructive method that has a strong history of applications in protein characterization. In this work we describe how Raman microscopy can be used as a fast and reliable method of tracking protein unfolding in the presence of a chemical denaturant. We have compared Raman spectroscopic data to the equivalent samples analyzed using fluorescence spectroscopy in order to validate the Raman approach. Calculations from both Raman and fluorescence unfolding curves of [D]50 values and Gibbs free energy correlate well with each other and more importantly agree with the values found in the literature for these proteins. In addition, 2D correlation analysis has been performed on both Raman and fluorescence data sets in order to allow further comparisons of the unfolding behavior indicated by each method. As many biopharmaceuticals are glycosylated in order to be functional, we compare the unfolding profiles of a protein (RNase A) and a glycoprotein (RNase B) as measured by Raman spectroscopy and discuss the implications that glycosylation has on the stability of the protein.

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Ribonucleases as Models for Understanding Protein Folding

Cited by 3 publications

References 119 publications

Dynamics of Disulfide-Bond Disruption and Formation in the Thermal Unfolding of Ribonuclease A

Dynamics of Disulfide-Bond Disruption and Formation in the Thermal Unfolding of Ribonuclease A

My 65 years in protein chemistry

Monitoring Guanidinium-Induced Structural Changes in Ribonuclease Proteins Using Raman Spectroscopy and 2D Correlation Analysis

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