Enthalpic and entropic contributions to actin stability: calorimetry, circular dichroism, and fluorescence study and effects of calcium.
The delta H associated with the thermal unfolding of G-actin has been determined by differential scanning calorimetry (DSC) to be 142 +/- 5 kcal/mol, with the Tm (melting temperature) at 57.2 +/- 0.5 degrees C, at pH 8.0 (heating rate 0.5 K/min). The transition is broad and cannot be treated as a single transition that mimics a two-state process, suggesting the existence of domains. Deconvolution is done to fit it into two quasi-independent two-state transitions. For F-actin, the transition is more cooperative, with a cooperative ratio (the ratio of van't Hoff enthalpy and calorimetric enthalpy) of 1.4, indicating intermonomer interaction. The delta H of the thermal unfolding of F-actin is 162 +/- 10 kcal/mol with a Tm at 67.0 +/- 0.5 degrees C. A state of G-actin similar to that of the heat-denatured form, designated D-actin, is obtained by removing tightly bound Ca2+ with EGTA. The DSC-detectable cooperative transition is completely lost when the free calcium concentration of the medium is 1 x 10(-11) M or lower, using a Ca2+/EGTA buffer system. However, circular dichroism (CD) shows that the helix content of actin, 32% in the G-form, is only partially reduced to 19% in this apo form. The CD spectrum and the helix content of the calcium-depleted actin are almost identical with those of the heat-denatured D form. This loss of 40% of the native helical content is irreversible in both cases. The remaining 60% of the native helical content cannot be further eliminated by heating to 95 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Bertazzon
A
A
Department of Biochemistry, University of Minnesota College of Biological Sciences, St. Paul 55108.
Tian
G H
GH
Lamblin
A
A
Tsong
T Y
TY
D000199:Actins; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002151:Calorimetry; D002942:Circular Dichroism; D004355:Drug Stability; D004533:Egtazic Acid; D005453:Fluorescence; D019791:Guanidine; D006146:Guanidines; D006358:Hot Temperature; D008297:Male; D009690:Nucleic Acid Conformation; D011489:Protein Denaturation; D011817:Rabbits; D013816:Thermodynamics; D014364:Tryptophan
D000199:Actins; D006146:Guanidines; D004533:Egtazic Acid; D014364:Tryptophan; D019791:Guanidine; D002118:Calcium