Abstract:
:Thermodynamic and structural evidence indicates that the DNA binding domains of lac repressor (lacI) exhibit significant conformational adaptability in operator binding, and that the marginally stable helix-turn-helix (HTH) recognition element is greatly stabilized by operator binding. Here we use circular dichroism at 222 nm to quantify the thermodynamics of the urea- and thermally induced unfolding of the marginally stable lacI HTH. Van't Hoff analysis of the two-state unfolding data, highly accurate because of the large transition breadth and experimental access to the temperature of maximum stability (T(S); 6-10 degrees C), yields standard-state thermodynamic functions (deltaG(o)(obs), deltaH(o)(obs), deltaS(o)(obs), deltaC(o)(P,obs)) over the temperature range 4-40 degrees C and urea concentration range 0 = C(3) = 6 M. For unfolding the HTH, deltaG(o)(obs) decreases linearly with increasing C(3) at all temperatures examined, which directly confirms the validity of the linear extrapolation method (LEM) to obtain the intrinsic stability of this protein. At 25 degrees C (pH 7.3 and 50 mM K(+)), both linear extrapolation and extrapolation via the local-bulk domain model (LBDM) to C(3) = 0 yield deltaG(o)(obs) = 1.23 +/- 0.05 kcal mol(-)(1), in agreement with direct measurement (1.24 +/- 0.30 kcal mol(-)(1)). Like deltaG(o)(obs), both deltaH(o)(obs) and deltaS(o)(obs) decrease linearly with increasing C(3); the derivatives with respect to C(3) of deltaG(o)(obs), deltaH(o)(obs) and TdeltaS(o)(obs) (in cal mol(-)(1) M(-)(1)) are -449 +/- 11, -661 +/- 90, and -203 +/- 91 at 25 degrees C, indicating that the effect of urea on deltaG(o)(obs) is primarily enthalpic. The deltaC(o)(P,obs) of unfolding (0.63 +/- 0.05 kcal mol(-)(1) K(-)(1)) is not detectibly dependent on C(3) or temperature. The urea m-value of the lacI HTH (-d deltaG(o)(obs),/dC(3) = 449 +/- 11 cal mol(-)(1) M(-)(1) at 25 degrees C) is independent of C(3) up to at least 6 M. Use of the LBDM to fit the C(3)-dependence of deltaG(o)(obs) yields the local-bulk partition coefficient for accumulation of urea at the protein surface exposed upon denaturation: K(P) = 1.103 +/- 0.002 at 25 degrees C. This partition coefficient is the same within uncertainty as those previously determined by LBDM analysis of osmometric data for solutions of urea and native (folded) bovine serum albumin, as well as LBDM analysis of the proportionality of m-values to changes in water accessible surface area upon protein unfolding. From the correspondence between values of K(P), we conclude that the average local urea concentration at both folded and unfolded protein surface exceeds the bulk by approximately 10% at 25 degrees C. The observed decrease in m-value for the lacI HTH with increasing temperature, together with the observed reductions in both deltaH(o)(obs) and deltaS(o)(obs) of unfolding with increasing urea concentration, demonstrate that K(P) for urea decreases with increasing temperature and that transfer of urea from the bulk solution to the local domain at the protein surface exposed on denaturation is enthalpically driven and entropically unfavorable.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Felitsky DJ,Record MT Jrdoi
10.1021/bi0270992subject
Has Abstractpub_date
2003-02-25 00:00:00pages
2202-17issue
7eissn
0006-2960issn
1520-4995journal_volume
42pub_type
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