[21]Protein conformational dynamics measured by hydrogen isotope exchange techniques

Gregory, Roger B.; Rosenberg, Andreas

doi:10.1016/0076-6879(86)31052-8

Cited by 59 publications

(41 citation statements)

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“…Deuterated buffers were prepared from a liophylized solution by adding D 2 O and adjusting the measured pH (pH*) with DCl. The pH* value was corrected using the equation pD ) pH* + 0.4 (Gregory & Rosenberg, 1986).…”

Section: Methodsmentioning

confidence: 99%

Scanning Calorimetry and Fourier-Transform Infrared Studies into the Thermal Stability of Cleaved Bacteriorhodopsin Systems

et al. 1996

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Differential scanning calorimetry and Fourier-transform infrared spectroscopy have been used to characterize the thermal stability of bacteriorhodopsin (BR) cleaved within different loops connecting the helical rods. The results are compared to those of the native protein. We show that the denaturation temperature and enthalpy of BR cleaved at peptide bond 71-72 or 155-156 are lower than those of the intact protein, and that these values become even lower for the BR cleaved at both peptide bonds. The effect of cleavage on the denaturation temperature and enthalpy values seems to be additive as has been previously suggested [Khan, T. W., Sturtevant, J. M., & Engelman, D. M. (1992) Biochemistry 31, 8829]. The thermal denaturation of all the samples was irreversible and scan-rate dependent. When cleaved at the 71-72 bond BR follows quantitatively the predictions of the two-state kinetic model at pH 9.5, with an activation energy of 374 kJ/mol, similar to that of native BR. Calorimetry experiments with different populations of intact and cleaved BR provide direct evidence for some intermolecular cooperativity upon denaturation. The denatured samples maintain a large proportion of R helices and structure, a fact which seems to be related to their low denaturation enthalpy as compared to that of water-soluble, globular proteins.Thermodynamic-data analysis and energetic characterization of the thermal stability of membrane proteins (SanchezRuiz & Mateo, 1987;Ruiz-Sanz et al., 1992) as well as that of several globular proteins (Sanchez-Ruiz et al., 1988;Conejero-Lara et al., 1991) cannot be undertaken because of their non-equilibrium, irreversible denaturation (SanchezRuiz, 1992). This irreversibility is usually due to nonequilibrium processes taking place on protein unfolding (Klibanov & Ahern, 1987). Even in this case differential scanning calorimetry (DSC) 1 can provide the denaturation enthalpy (∆H) and temperature (T m ) values of the transition for a given scan rate. Thus T m can be used as an operative parameter, at least in comparative, relative terms, to characterize the thermal stability of a protein which unfolds under non-equilibrium conditions. Fourier-transform infrared spectroscopy (FTIR) is a very suitable technique to complement protein DSC studies, particularly in the case of membrane proteins, since it provides structural information concerning the native and denatured states and can also be used to observe the denaturation process itself (Surewicz & Mantsch, 1988;Surewicz et al., 1993;Arrondo et al., 1993;Jackson & Mantsch, 1995).Bacteriorhodopsin (BR), the only protein present in the purple membrane of Halobacterium salinarium, is one of the best known and well-studied intrinsic membrane proteins, both from a structural and a functional point of view (Henderson et al., 1990;Rothschild, 1992;Lanyi, 1993;Grigorieff et al., 1996). Previous denaturation studies carried out by DSC and spectroscopic techniques have thrown light on the importance of several structural features on BR's stability, ...

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Section: Methodsmentioning

confidence: 99%

Scanning Calorimetry and Fourier-Transform Infrared Studies into the Thermal Stability of Cleaved Bacteriorhodopsin Systems

et al. 1996

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“…However, dynamic solvent accessibility based on noncooperative, small-scale protein motions which can allow NHsolvent contact cannot be excluded. The mobile defect and expansile cavity and channel formation models (e.g., see Woodward et al, 1982;Englander and Kallenbach, 1984;Gregory and Rosenberg, 1986) could be classified under the native-state mechanism for exchange. Although the present study of LbCO cannot exclude exchange by such mechanisms, our data, under native protein conditions, are adequately described by the simpler model of cooperative local unfolding.…”

Section: Lbco Packing and Stability With The Possible Exception Ofmentioning

confidence: 99%

“…The requirement for significant protection from exchange is that an amide participates in the hydrogen bonded structure and is present in a hydrophobic environment shielded from solvent. Global or local fluctuations have the effect of partial rearrangement of secondary andor tertiary structural elements and hydrogen-bond rupture, leading to amide-proton exchange (for reviews see Hvidt and Nielsen, 1966;Woodward et al, 1982;Englander and Kallenbach, 1984;Gregory and Rosenberg, 1986). In addition, H/D exchange can provide insight into transient folding intermediates, compact non-native protein states (e.g.…”

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confidence: 99%

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Hydrogen Exchange in the Carbon Monoxide Complex of Soybean Leghemoglobin

Morikis¹,

Wright²

1996

European Journal of Biochemistry

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Hydrogen/deuterium exchange rates for individual amide protons have been measured for the carbon monoxide complex of soybean leghemoglobin. Fast two-dimensional NOESY experiments were performed, with 5.2-min data-collection time for each spectrum, which made possible the measurement of NOE cross-peaks of relatively rapidly exchanging amide protons at early time points. Exchange rates were measured for 61 backbone amides, and protection factors were calculated to provide information on the packing and local stability of the protein. The data are consistent with the presence of transient cooperative local unfolding of helical segments. The B-, E-, G-and H-helices have extensive regions of slow-, medium-and fast-exchanging amide protons. For each of these helices, there is a progressive decrease in protection on moving from the helix center to the termini. This is consistent with a stable helix center, with dynamic fraying at the ends. Amide exchange from the A-helix and C-helix is rapid except in small local regions. The F-helix, which is located on the proximal side of the heme pocket and

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“…Within the native structure of proteins, amide hydrogen exchange rates can vary by several orders of magnitude and these rates are sensitive to changes in protein conformation (reviewed by Englander & Kallenbach, 1983;Gregory & Rosenberg, 1986).…”

mentioning

confidence: 99%

Topographic study of arrestin using differential chemical modifications and hydrogen/deuterium exchange

Ohguro¹,

Palczewski²,

Walsh³

et al. 1994

Protein Science

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Arrestin is involved in the quenching of phototransduction by binding to photoactivated and phosphorylated rhodopsin (P-Rho*). To study its conformational changes and regions interacting with P-Rho*, arrestin was subjected to (1) differential acetylation at lysine residues in the presence and absence of P-Rho*, and (2) amide hydrogen/ deuterium exchange. Labeled protein was proteolysed and analyzed by mass spectrometry. Three Lys residues, 28, 176, and 21 I, were protected from acetylation in native arrestin, although they were not located in regions exhibiting slow amide hydrogen exchange rates. The presence of P-Rho* protected lysine 201 from acetylation and partially protected 14 other lysyl residues, including (2, 5 ) , (163, 166, 167), (232, 235, 236, 238), (267, 276), (298, 300), and 367, where parentheses indicate lysine residues found within the same peptide. In contrast, in the C-terminal region of arrestin, lysyl residues (386, 392, 395) were more exposed upon binding to P-Rho*. These data allowed us to identify functional regions in the arrestin molecule.

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[21]Protein conformational dynamics measured by hydrogen isotope exchange techniques

Cited by 59 publications

References 100 publications

Scanning Calorimetry and Fourier-Transform Infrared Studies into the Thermal Stability of Cleaved Bacteriorhodopsin Systems

Scanning Calorimetry and Fourier-Transform Infrared Studies into the Thermal Stability of Cleaved Bacteriorhodopsin Systems

Hydrogen Exchange in the Carbon Monoxide Complex of Soybean Leghemoglobin

Topographic study of arrestin using differential chemical modifications and hydrogen/deuterium exchange

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