pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1

Pace, C. Nick; Laurents, Douglas V.; Thomson, James A.

doi:10.1021/bi00462a019

Cited by 362 publications

(342 citation statements)

References 52 publications

Supporting

Mentioning

299

Contrasting

Order By: Relevance

“…For OMTKY3, A a in urea is 0.19, while in GuHCl it is 0.28. For other proteins reported in the literature, values of A a in the two denaturants are the same (Pace et al, 1990). In the case of OMTKY3, Aa(GuHC1) may be artifactually high because of the stabilizing effects discussed previously.…”

Section: Discussionmentioning

confidence: 64%

Thermodynamics of unfolding for turkey ovomucoid third domain: Thermal and chemical denaturation

1993

View full text Add to dashboard Cite

We have used thermal and chemical denaturation to characterize the thermodynamics of unfolding for turkey ovomucoid third domain (OMTKY3). Thermal denaturation was monitored spectroscopically at a number of wavelengths and data were subjected to van't Hoff analysis; at pH 2.0, the midpoint of denaturation (T,) occurs at 58.6 -t 0.4 "C and the enthalpy of unfolding at this temperature (AH,) is 40.8 -t 0.3 kcal/mol. When T, was perturbed by varying pH and denaturant concentration, the resulting plots of AH, versus T, yield a mean value of 590 5 120 cal/(mol .K) for the change in heat capacity upon unfolding (AC,). A global fit of the same data to a n equation that includes the temperature dependence for the enthalpy of unfolding yielded a value of 640 k 110 cal/(mol .K). We also performed a variation of the linear extrapolation method described by Pace and Laurents, which is an independent method for determining ACp (Pace, C.N. & Laurents, D., 1989, Biochemistry 28, 2520-2525). First, OMTKY3 was thermally denatured in the presence of a variety of denaturant concentrations. Linear extrapolations were then made from isothermal slices through the transition region of the denaturation curves. When extrapolated free energies of unfolding (AG,) were plotted versus temperature, the resulting curve appeared linear; therefore, AC, could not be determined. However, the data for AG, versus denaturant concentration are linear over an extraordinarily wide range of concentrations. Moreover, extrapolated values of AG, in urea are identical to values measured directly.Keywords: heat capacity; linear extrapolation method; protein folding; van't Hoff analysis One facet of "the protein folding problem" is understanding how proteins maintain their unique native conformations. While both the energetics and conformation of the native structure are clearly dictated by the primary amino acid sequence, the contributions of individual residues are at present ill-defined. Comprehensive studies of such contributions can most easily be addressed in small proteins, such as ovomucoid third domain (56 amino acid residues, MW = 6.1 kDa). Our ultimate goal is to quantitatively describe the contribution of every amino acid residue to theglobal stability of ovomucoid third domain. Key information for these studies is knowledge of the free energy of unfolding (AG,) over a wide range of experimental conditions. AG, can be directly measured over only a narrow temperature range but, for a two-state process, can be predicted at any temperature from the modified Gibbs-Helmholtz equation:AG, = AH,-(l -T/T,)

show abstract

Section: Discussionmentioning

confidence: 64%

Thermodynamics of unfolding for turkey ovomucoid third domain: Thermal and chemical denaturation

1993

View full text Add to dashboard Cite

show abstract

“…Direct measurements of ∆G unf versus pH by urea or GdmCl unfolding have been made for a few proteins such as RNase A (36,37), and they confirm that there is a large decrease in ∆G unf at acid pH. The pK a values of all the carboxylate residues of T4 lysozyme have been measured by NMR and used to predict the pH dependence of ∆G unf.…”

Section: Using C M Values To Measure Relative Stability Of Mutantsmentioning

confidence: 89%

Putative Interhelix Ion Pairs Involved in the Stability of Myoglobin

1999

View full text Add to dashboard Cite

An earlier theoretical study predicted that specific ion pair interactions between neighboring helices should be important in stabilizing myoglobin. To measure these interactions in sperm whale myoglobin, single mutations were made to disrupt them. To obtain reliable ∆G values, conditions were found in which the urea induced unfolding of holomyoglobin is reversible and two-state. The cyanomet form of myoglobin satisfies this condition at pH 5, 25°C. The unfolding curves monitored by far-UV CD and Soret absorbance are superimposable and reversible. None of the putative ion pairs studied here makes a large contribution to the stability of native myoglobin. The protein stability does decrease somewhat between 0 and 0.1 M NaCl, however, indicating that electrostatic interactions contribute favorably to myoglobin stability at pH 5.0. A previous mutational study indicated that the net positive charge of the A[B]GH subdomain of myoglobin is an important factor affecting the stability of the pH 4 folding intermediate and potential ion pairs within the subdomain do not contribute significantly to its stability. One of the assumptions made in that study is tested here: replacement of either positively or negatively charged residues outside the A[B]GH subdomain has no significant effect on the stability of the pH 4 molten globule.Pairs of oppositely charged residues on adjacent helices are predicted to make strong electrostatic interactions in sperm whale Mb 1 (1). These interactions are predicted to be screened by counterions although, if they are present as salt bridges (hydrogen-bonded ion pairs), these are not found to be very sensitive to counterion screening, in peptide helices (2, 3). In either case, if there are any strong pairwise interactions between oppositely charged residues on adjacent helices, they are likely to be important in determining the folding pathway of apoMb. We undertook to measure them, to find out if any changes in stability are indeed caused by pairwise interactions, and to determine how the stability of Mb is affected by counterion screening.Our procedure is to substitute by alanine at least one member of each potential ion pair and to determine the resulting change (∆∆G) in its free energy of unfolding (∆G unf ). If replacing one residue gives a significant change in ∆G unf , then the other residue of the pair is substituted with alanine and ∆∆G is measured to find out if the second mutation causes an equally large change, as expected if there is a pairwise interaction. The test for a pairwise interaction is essential when mutation of one member of a pair gives a large change in stability because other effects, such as steric, hydrophobic, or helix propensity effects, may be responsible for the observed change. To obtain reliable ∆∆G values, the folding transition must be reversible and two-state. We searched for conditions in which the urea-induced unfolding of holoMb satisfies this condition. GdmCl is unsatisfactory for this purpose because high Cl -concentrations stabilize a fold...

show abstract

“…The two extreme examples correspond to LCI (in which urea is unable to quantitatively unfold LCI) and TAP, where urea is more potent than GdnHCl. The clear difference of potency between urea and GdnHCl has been extensively reported and can be attributed to the differential mode of action between these denaturants, although the detailed mechanisms are not yet fully understood (15,11,16,17). It is known that both denaturants disrupt noncovalent interactions that stabilize the native fold (18), mainly hydrophobic interactions, by causing water to become a better solvent for nonpolar amino acids.…”

Section: Table Imentioning

confidence: 99%

The Unfolding Pathway of Leech Carboxypeptidase Inhibitor

Salamanca¹,

Villegas²,

Vendrell³

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

The unfolding and denaturation curves of leech carboxypeptidase inhibitor (LCI) were elucidated using the technique of disulfide scrambling. In the presence of thiol initiator and denaturant, the native LCI denatures by shuffling its native disulfide bonds and transforms into a mixture of scrambled species. 9 of 104 possible scrambled isomers of LCI, amounting to 90% of total denatured LCI, can be distinguished. The denaturation curve that plots the fraction of native LCI converted into scrambled isomers upon increasing concentrations of denaturant shows that the concentration of guanidine thiocyanate and guanidine hydrochloride required to reach 50% of denaturation is 2.4 and 3.6 M, respectively. In contrast, native LCI is resistant to urea denaturation even at high concentration (8 M). The LCI unfolding pathway was defined based on the evolution of the relative concentration of scrambled isoforms of LCI upon denaturation. Two populations of scrambled species suffer variations along the unfolding pathway. One accumulates as intermediates under strong denaturing conditions and corresponds to open or relaxed structures, among which the beads-form isomer is found. The other population shows an inverse correlation between their relative abundances and the denaturing conditions and should have another kind of non-native structure that is more compact than the unfolded state. The rate constants of unfolding of LCI are low when compared with other disulfide-containing proteins. Overall, the results presented in this study show that LCI, a molecule with potential biotechnological applications, has slow kinetics of unfolding and is highly stable.

show abstract

pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1

Cited by 362 publications

References 52 publications

Thermodynamics of unfolding for turkey ovomucoid third domain: Thermal and chemical denaturation

Thermodynamics of unfolding for turkey ovomucoid third domain: Thermal and chemical denaturation

Putative Interhelix Ion Pairs Involved in the Stability of Myoglobin

The Unfolding Pathway of Leech Carboxypeptidase Inhibitor

Contact Info

Product

Resources

About