PK Changes of Ionizable Reporter Groups as an Index of Conformational Changes in Proteins. A Study of Fluorescein-Labelled Ribonuclease A

Garel, Jean‐Renaud

doi:10.1111/j.1432-1033.1976.tb10968.x

Cited by 45 publications

(30 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Oligonucleotides were synthesized by Operon. Bovine RNase A was obtained from Sigma (grade XIIA) and further purified by the procedure of Garel (1976). Reduced and oxidized glutathione, 2',3'-cCMP (cyclic cytidine monophosphate), and IPTG were obtained from Sigma and Boehringer-Mannheim, respectively.…”

Section: Methodsmentioning

confidence: 99%

Cis proline mutants of ribonuclease A. I. thermal stability

Schultz

Baldwin

1992

Protein Science

View full text Add to dashboard Cite

A chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol. 185, 60-89). The expressed protein, which contains an additional N-terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A. The expressed protein accumulates in inclusion bodies and has scrambled disulfide bonds; the native disulfide bonds are regenerated during purification. Site-directed mutations have been made at each of the two cis proline residues, 93 and 114, and a double mutant has been made. In contrast to results reported for replacement of trans proline residues, replacement of either cis proline is strongly destabilizing. Thermal unfolding experiments on four single mutants give AT,,, e 10 "C and AAGo (apparent) = 2-3 kcal/mol. The reason is that either the substituted amino acid goes in cis, and cis H trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state, or else there is a conformational change, which by itself is destabilizing relative to the wild-type conformation, that allows the substituted amino acid to form a trans peptide bond.

show abstract

Section: Methodsmentioning

confidence: 99%

Cis proline mutants of ribonuclease A. I. thermal stability

Schultz

Baldwin

1992

Protein Science

View full text Add to dashboard Cite

show abstract

“…The absorbance of micellar solutions of FTC-HSA was kept between 0.06 -0.12 at 495 nm. The FTC/HSA molar ratio was 0.65 ( E = 72.1 mM-' cm-' at pH > 8.1; Garel, 1976).…”

Section: Chemical Modifications Of Albuminmentioning

confidence: 99%

Ligand binding at membrane mimetic interfaces

Desfosses¹,

Cittanova²,

Urbach³

et al. 1991

European Journal of Biochemistry

View full text Add to dashboard Cite

The behaviour of human serum albumin in the presence of three chemically distinct ligands: oxyphenylbutazone, dansylsarcosine and hemin, has been compared in buffer and in reverse micelles of isooctane, water, and either sodium bis(2-ethylhexyl)sulfosuccinate or hexadecyl trimethylammonium bromide, systems selected to mimic the membrane-water interface. Upon micellar incorporation, the dansylsarcosine-albumin complex dissociated, as evidenced by fluorescence emission spectroscopy (red shift from 485 nm to 570 nm) and by fluorescence polarization measurements. In contrast, the hemin-albumin complex remained stable in reverse micelles, as judged from the Soret absorption band at 408 nm and the molar absorption coefficient of 8.4 x lo4 M -' cm-'. The oxyphenylbutazone to albumin binding curves reveal that while the association constant remained unchanged ( K , 1.0 x lo5 M-'), only a fraction of the albumin molecules present reacted with the ligand. The results were unaffected by the nature and the concentration of the surfactant.These findings can be interpreted in the light of conformational changes induced in human serum albumin by the large micellar inner surface area. The blue shift of the fluorescence emission maximum from 344nm in buffer to 327 nm in sodium bis(2-ethylhexyl)sulfosuccinate micelles and the lesser reactivity/accessibility of the fluorophore to oxidation by N-bromosuccinimide, indicate perturbations of the sole tryptophan-214 microenvironment. However, the distance between the indole residue and tyrosine-41 1 does not seem substantially modified by the 15% decrease affecting the CI helices of the albumin molecule. It is proposed that the results reported herein reflect the interactions of albumin with a membrane-like interface which generates two protein subpopulations differing in their membrane-surface and ligand affinities. Overall and local conformational changes, originating from this surface-induced effect, may thus constitute a ligand-release facilitating mechanism acting at cellular membrane levels.

show abstract

“…3, the results of other double-jump experiments performed with RNase A (5, 6, 10) are plotted together with those obtained for the slower phase in the unfolding of nitrated RNase A. Both sets of data fit the same Arrhenius line, corresponding to an activation enthalpy of 18 kcal/mol and suggesting that they pertain to the same process. Also, the results given in Fig.…”

Section: Kinetics Of Proline Cis-trans Isomerization Of a Modelmentioning

confidence: 85%

“…MATERIALS AND METHODS RNase A was obtained from Sigma (lot 95 G 8147) and was purified by ion-exchange chromatography (18). Tetranitromethane was from Aldrich, and guanidinium hydrochloride (Gdn-HCI) was from Carlo Erba.…”

mentioning

confidence: 99%

Evidence for involvement of proline cis-trans isomerization in the slow unfolding reaction of RNase A.

Garel¹

1980

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The three exposed tyrosines of RNase A have been converted to nitrotyrosines by reaction with tetranitromethane, and the changes in the ionization properties of these nitrotyrosines have been used to follow the kinetics of unfolding of the nitrated protein. It is found that the nitrotyrosines not only are sensitive to the overall disruption of the protein structure, which occurs in a faster reaction, but also serve as reporter groups for the slower reaction which takes place in the unfolded state. This slower reaction corresponds to the formation of the slow-refolding species of the unfolded protein. The kinetic properties of the slower reaction-guanidine-dependence of the rate and activation enthalpy-are similar to those of the proline cis-trans isomerization in a model peptide determined in the same conditions. It is concluded that proline cis-trans isomerization is indeed the rate-limiting factor for the formation of the slow-refolding species. Because the influence of proline cis-trans isomerization on the properties of the nitrotyrosines in the unfolded protein is probably due to a local effect, it is suggested that most of the optical changes observed during this slow unfolding reaction arise from the effect of the cis-trans isomerization of the Asn"13-Pro"'4 bond on the properties of nitrotyrosine 115.Over a wide range of conditions, the reactions of unfolding and refolding of RNase A can be described by a three-species mechanism:slow fast U1 ±IU2± zN in which N is the native protein and Ui and U2 are two different, unfolded, optically indistinguishable species in slow equilibrium (1-6). The fast-refolding species, U2, represents about 20% of the protein molecules and can fold up several hundred times faster than the slow-refolding species, Ui (4,5). The difference between U1 and U2 probably lies in the configuration of the polypeptide chain, and, more specifically, it has been proposed that U1 and U2 differ only in the cis-trans isomers of their four proline residues (6). Indeed, proline-containing peptides exist as a mixture of cis and trans isomers, and the interconversion between cis and trans is a slow process (5-8). The fast-refolding species, U2, would have all its proline residues in the same cis or trans state as in N, whereas the slow-refolding species, U1, would correspond to molecules with at least one proline residue in a "non-native" cis or trans state (6).This explanation of the slow reactions of unfolding and refolding in RNase A is esthetically appealing because of its simplicity; it is also difficult to test, mainly because there are few ways of distinguishing the cis and trans states of a given proline residue in a polypeptide chain while it is folding. Two recent pieces of evidence suggest that proline cis-trans isomerization is indeed involved in the slow folding reaction of simple proteins. First, a form of parvalbumin that does not contain proline does not show a slow folding reaction (9). Second, the slow unfolding reaction of RNase A is acid-catalyzed (10), as is the inter...

show abstract

PK Changes of Ionizable Reporter Groups as an Index of Conformational Changes in Proteins. A Study of Fluorescein-Labelled Ribonuclease A

Cited by 45 publications

References 25 publications

Cis proline mutants of ribonuclease A. I. thermal stability

Cis proline mutants of ribonuclease A. I. thermal stability

Ligand binding at membrane mimetic interfaces

Evidence for involvement of proline cis-trans isomerization in the slow unfolding reaction of RNase A.

Contact Info

Product

Resources

About