Thermodynamics of the denaturation of ribonuclease by guanidine hydrochloride

Salahuddin, A; Tanford, Charles

doi:10.1021/bi00808a007

Cited by 90 publications

(35 citation statements)

References 13 publications

(18 reference statements)

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“…The enthalpy of denaturation of apoA-I (2.4 cal/g) is small in comparison with other globular proteins (9,11,13) and results in AG (370) = 2.4 kcal/mol. This AG is strikingly less than that obtained for other globular proteins by calorimetry (8-14 kcal/ mol) (9) or other methods (9-16 kcal/mol) (15)(16)(17) at similar temperatures. This means that the free energy of stabilization of the native structure of apoA-I at 370 is much lower than that of other water-soluble proteins, such as ribonuclease or lysozyme.…”

contrasting

confidence: 59%

Apoprotein stability and lipid-protein interactions in human plasma high density lipoproteins.

Tall¹,

Small²,

Shipley³

et al. 1975

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

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Temperature-dependent conformational changes of the principal apoprotein of human plasma high density lipoprotein (HDL), apoA-I, have been studied in the isolated apoprotein, in complexes of apoprotein with phospholipid, and in intact HDL. Differential scanning calorimetry shows that in solution apoA-I undergoes a reversible, twostate thermal denaturation (midpoint temperature 540). The enthalpy (2.4 cal/g) (10.0 J/g) and specific heat change (0.08 cal/°C per g) (0.33 J/°C per g) associated with the denaturation were used to calculate the free energy difference (AG) between native and unfolded apoA-I at 37°. AG (2.4 kcal/ mol) (10.0 kJ/mol) is less than that of other globular proteins (typically 8-14 kcal/mol) (33-59 kJ/mol), indicating that at 370 native apoA-I has a loosely folded conformation. Turbidity studies show that apoA-I is able to solubilize phospholipidin its native but not in its denatured form. Mixtures of apo-HDL (the total apoprotein of HDL) or apoA-I with dimyristoyl lecithin show a thermal transition at about 850 n-ot present in the lecithin or the apoprotein alone, which indicates that the native conformation of the apoprotein is stabilized by phospholipid. Scanning calorimetry of intact HDL shows a high-temperature endotherm associated with disruption of the HDL particle, su esting that in HDL the conformation of apoA-I is also stabilized by interaction with lipid. The loosely folded conformation of native, uncomplexed apoA-I may be specially adapted to the binding of lipid, since this process may involve both hydrophobic sites on the surface of the protein and concealed apolar amino acid residues that are exposed by a cooperative, low energy unfolding process.A knowledge of the structure and thermodynamic stability of serum lipoproteins is important to an understanding of their metabolism and role in diseases such as atherosclerosis. The human plasma high density lipoproteins (HDL) are spherical particles (1, 2) consisting of about 50% protein, 22% phospholipids, 3% free cholesterol, 14% cholesterol esters, and 8% triglycerides (3). The proteins (apo-HDL) include approximately 60-65% apoA-I [molecular weight (Mr) 28,331], 30% apoA-2 (Mr 17,380) and 5-10% low Mr C-peptides (4, 5). The proteins and polar head groups of phospholipids occupy the surface of the lipoprotein particle while the apolar lipid moieties form a hydrophobic core (1, 2). Further, the conformations of the apoproteins of HDL appear to be stabilized by interaction with lipids (3, 4). In the present study, we show that the principal apoprotein of human HDL, apoA-I, undergoes a reversible temperaturedependent conformational change with unusual thermodynamic properties. A comparison with other water-soluble proteins suggests a loosely folded structure of the native apo- Fig. la and b). From the DSC thermogram, it is possible to measure both the calorimetric enthalpy of the transition in cal/g (AHcal) (1 cal = 4.184 J) from the area under the curve, and the effective enthalpy in cal/mol (AHvH) using an expression der...

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contrasting

confidence: 59%

Apoprotein stability and lipid-protein interactions in human plasma high density lipoproteins.

Tall¹,

Small²,

Shipley³

et al. 1975

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

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“…A different measurement of the refolding process is provided by the change in absorbance at 287 nm, which accompanies the burial of three tyrosine groups in native RNase A and monitors the exclusion of water from the interior of the protein. Refolding is studied in jumps across the entire transition zone, so that only unfolded species are present in the initial conditions and, since the reaction is completely reversible (16), only native RNase A is present in the final conditions.…”

mentioning

confidence: 99%

Guanidine-unfolded state of ribonuclease A contains both fast- and slow-refolding species.

Garel¹,

Nall²,

Baldwin³

1976

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

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The kinetics of the refolding reaction of ribonuclease A from high concentrations of guanidine hydrochloride or urea are biphasic, and show two refolding reactions whose rates differ 450-fold at pH 5.8 and 250. Measurements of cytidine 2'-phosphate binding during refolding, after stopped-flow dilution of guanidine hydrochloride (Gdn-HCl) or urea, show that functional bovine pancreatic ribonuclease A (RNase A; ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) is formed in both the fast and slow phases of the refolding process. We conclude that the guanidine-unfolded state of RNase A is an equilibrium mixture of fast-and slow-refolding species, as was found previously for the heat-unfolded state at low pH. The fraction of the fast-refolding species in guanidine or ureaunfolded RNase A is the same as that in the heat-unfolded protein at pH 2.Previous work has shown that the fast-refolding species disappears as the pH is raised from 3 to 5 for heat-unfolded RNase A. This pH effect is not present in refolding from concentrated GdnHCl solutions: the same proportion of the fast-refolding species is found from pH 2 to pH 6, and also from 2 M to 6 M Gdn-HCI at pH 5.8. We conclude that the same proportion of the fast-refolding species is present at equilibrium whenever the residual structure in unfolded RNase A is reduced to a low level, and that the structural difference between the fast-refolding and slow-refolding species of RNase A lies in the configuration of the random coil polypeptide chain.The observed rates of protein folding reactions are many orders of magnitude faster than predicted from a purely random search of all possible configurations for a random coil polypeptide chain devoid of structuret. A possible explanation for this difference is that an unfolded protein is not a random coil. The elements of residual structure play a crucial role in directing the course of protein folding, and thus considerably restrict the possible pathways (1) There seems to be a contradiction between the two proposals: (a) that a guanidine-unfolded protein behaves as a homogeneous random chain and (b) that this same unfolded state still possesses some elements of residual structure which increase the rate and determine the pathway(s) of folding. However, these proposals are compatible if the residual structure, important for the folding process, is not present as such in the unfolded state, but is rapidly formed after refolding is initiated. For instance, formation of a-helical segments could take place within the dead time of stopped-flow measurements (6) and limit the possible pathways for refolding, if the a-helical segments are stable under the conditions in which refolding is initiated, without a requirement of prior slow steps in refolding to provide a stabilizing environment.Studies of the refolding of heat-unfolded RNase A (7-11) have shown that the heat-unfolded state does not behave as a single species in refolding. Instead, distinct fast-refolding and slow-refolding species of the heat-unfold...

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“…Since there is no a priori correlation between the magnitude of a chemical shift change and the magnitude of the conformational change producing it, we cannot characterise these intermediates more precisely than this at the present time. The conflict between these results and the optical spectroscopic studies of Salahuddin and Tanford [7] is probably only apparent; it is likely that the NMR results reflect only small deviations from "two-state" behaviour. However, these small deviations are of considerable importance since they open the way to a description of the pathway of unfolding.…”

Section: Resultsmentioning

confidence: 71%

“…In the unfolded protein, the magnetic environment of all four histidine residues is the same, so the four C2 protons give rise to a single resonance line. Since separate and moderately sharp resonances are seen for the native and unfolded states, the exchange between these states must be slow on the NMR time-scale; in this case the rate of exchange must be less than about 70 set-l. Salahuddin and Tanford [7] find approx. 0.002 see-l for the overall denaturation at 2.88 M GuHCl.…”

Section: Resultsmentioning

confidence: 96%

“…Only rarely have such intermediates been detected by the methods (such as optical rotation, ultraviolet absorption) commonly used to study denaturation [ 1, 21. In the case of ribonuclease, there is some evidence for the existence of intermediates in thermal unfolding [ 1,3], notably from the elegant kinetic experiments of Tsong et al [4-61, but Salahuddin and Tanford [7] concluded that the guanidine hydrochloride denaturation is a "two-state" process.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

NMR Studies of the unfolding of ribonuclease by guanidine hydrochloride. Evidence for intermediate states

Benz

Roberts

1973

FEBS Letters

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Thermodynamics of the denaturation of ribonuclease by guanidine hydrochloride

Cited by 90 publications

References 13 publications

Apoprotein stability and lipid-protein interactions in human plasma high density lipoproteins.

Apoprotein stability and lipid-protein interactions in human plasma high density lipoproteins.

Guanidine-unfolded state of ribonuclease A contains both fast- and slow-refolding species.

NMR Studies of the unfolding of ribonuclease by guanidine hydrochloride. Evidence for intermediate states

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