The Ionization Constant of Deuterium Oxide from 5 to 50°

Covington, A. K.; Robinson, R. A.; Bates, Roger G.

doi:10.1021/j100884a011

Cited by 183 publications

(143 citation statements)

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“…KD was taken as 10 Ϫ15.05 (62). Solution nonideality effects in water dissociation (pKD) and water chemical activity (aD20) were taken into account as follows.…”

Section: Methodsmentioning

confidence: 99%

Urea, but not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide group

Lim

Rösgen

Englander

2009

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

354

370

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The mechanism by which urea and guanidinium destabilize protein structure is controversial. We tested the possibility that these denaturants form hydrogen bonds with peptide groups by measuring their ability to block acid-and base-catalyzed peptide hydrogen exchange. The peptide hydrogen bonding found appears sufficient to explain the thermodynamic denaturing effect of urea. Results for guanidinium, however, are contrary to the expectation that it might H-bond. Evidently, urea and guanidinium, although structurally similar, denature proteins by different mechanisms.denaturation ͉ hydrogen exchange ͉ osmolyte ͉ solvation B ecause of its fundamental importance, the study of protein molecular stability has engaged the attention of protein chemists for the last 100 years (1, 2). Experimental and theoretical investigations of protein stability have often focused on small-molecule osmolytes that can be used to modulate stability in vitro (3-6) and are used by virtually all organisms to counter biochemical stress in vivo (7). The mechanism of osmolyte action continues to be controversial. Opposing positions favor either a direct interaction between protein and osmolyte (8-11), such as hydrogen bonding, or an indirect effect mediated by the alteration of water structure (12, 13), or a mixture of both (14)(15)(16)(17)(18)(19). It has been difficult to distinguish between these direct and indirect models because the osmolyte-protein interaction is so weak.It is a thermodynamic truism (20-22) that the concentration of destabilizing osmolytes must be enriched relative to water molecules in the vicinity of the protein surface that becomes newly exposed upon unfolding (preferential interaction). In a thermodynamic sense, denaturing osmolytes ''bind'' selectively to the increased surface of unfolded proteins and thus bias the folded/ unfolded equilibrium toward denaturation. Similarly, stabilizing osmolytes must be preferentially excluded from the protein surface. This is true independently of the physical mechanism of the ''binding'' or ''antibinding'' interaction. Thermodynamically based studies directed at the binding/antibinding problem have been designed to measure the transfer free energy of the various component groups of proteins between water and osmolyte solutions. Results show that the main-chain peptide group makes the largest contribution to the energetics, accounting for Ϸ80% of the effect of urea and of strong stabilizers such as trimethyl amine N-oxide (TMAO) (23). Similar determinations for guanidinium are not yet available, but it seems clear that guanidinium also favorably interacts with the peptide group (8,11,24).To search for the mechanistic basis of thermodynamic destabilization due to urea and guanidinium, we tested the possibility that they exert their denaturing effect through peptide group hydrogen bonding. This can be done experimentally by the straightforward measurement of acid-and base-catalyzed hydrogen exchange (HX) in a small-molecule peptide model. Osmolyte that is H-bonded to the peptide NH ...

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“…KD was taken as 10 Ϫ15.05 (62). Solution nonideality effects in water dissociation (pKD) and water chemical activity (aD20) were taken into account as follows.…”

Section: Methodsmentioning

confidence: 99%

Urea, but not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide group

Lim

Rösgen

Englander

2009

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

354

370

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“…(2a), eliminates the need to separately compute [OD − ] as a function of temperature. Alternatively, one can use 3 kcal/mol for the activation energy of k B , ref and separately calculate specific base concentration (OD − or OH − ) from known pH and the equations provided by Covington et al 36 for solvent pK a . The two approaches give very similar results over a wide temperature range.…”

Section: Temperature Dependencementioning

confidence: 99%

Primary structure effects on peptide group hydrogen exchange

et al. 1993

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The rate of exchange of peptide group NH hydrogens with the hydrogens of aqueous solvent is sensitive to neighboring side chains. To evaluate the effects of protein side chains, all 20 naturally occurring amino acids were studied using dipeptide models. Both inductive and steric blocking effects are apparent. The additivity of nearest-neighbor blocking and inductive effects was tested in oligo-and polypeptides and, surprisingly, confirmed. Reference rates for alanine-containing peptides were determined and effects of temperature considered. These results provide the information necessary to evaluate measured protein NH to ND exchange rates by comparing them with rates to be expected for the same amino acid sequence is unstructured oligo-and polypeptides. The application of this approach to protein studies is discussed.

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“…Two sets of samples were used. In one set exchange was followed at selected pH values between pH 5.2 and pH 8.1 at a constant temperature of 298 K. For the second set exchange was followed at temperatures between 280 and 300 K. At each temperature, pH was adjusted to give a constant [OD-] of 1.55 X * 3.98 X IO-' M (Glascoe & Long, 1960;Covington et al, 1966). At the applied pH-and temperature ranges, ACBP is known to maintain a native and folded structure.…”

Section: Exchange Measrtrementsmentioning

confidence: 99%

Mapping the lifetimes of local opening events in a native state protein

Kragelund¹,

Heinemann²,

Knudsen³

et al. 1998

Protein Science

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The rate constants for the processes that lead to local opening and closing of the structures around hydrogen bonds in native proteins have been determined for most of the secondary structure hydrogen bonds in the four-helix protein acyl coenzyme A binding protein. In an analysis that combines these results with the energies of activation of the opening processes and the stability of the local structures, three groups of residues in the protein structure have been identified. In one group, the structures around the hydrogen bonds have frequent openings, every 600 to 1,500 s, and long lifetimes in the open state, around 1 s. In another group of local structures, the local opening is a very rare event that takes place only every 15 to 60 h. For these the lifetime in the open state is also around 1 s. The majority of local structures have lifetimes between 2,000 and 20,000 s and relatively short lifetimes of the open state in the range between 30 and 400 ms. Mapping of these groups of amides to the tertiary structure shows that the openings of the local structures are not cooperative at native conditions, and they rarely if ever lead to global unfolding. The results suggest a mechanism of hydrogen exchange by progressive local openings.

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The Ionization Constant of Deuterium Oxide from 5 to 50°

Cited by 183 publications

References 1 publication

Urea, but not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide group

Urea, but not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide group

Primary structure effects on peptide group hydrogen exchange

Mapping the lifetimes of local opening events in a native state protein

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