Unfolding energetics of G-alpha-actin: a discrete intermediate can be re-folded to the native state by CCT.
Nascent actin requires interactions with the highly conserved and essential eukaryotic chaperonin-containing TCP-1 (CCT) for its correct folding to the native state in vivo. Biochemical and structural analysis of the interaction between actin and CCT has been studied extensively but the underlying energetics and kinetics of the CCT-dependent actin folding process are not understood. We investigated the unfolding and folding pathways of actin, using stopped flow fluorescence and biochemical techniques. By using very low concentrations of actin, taking account of temperature and ATP concentration dependences we were able to determine accurately the activation energy of unfolding to a stable intermediate, I(3). Use of the fluorescent calcium chelator Quin-2 and consideration of the ATP concentration dependence on the unfolding rate has allowed the intrinsic kinetics to be linked to the accepted reaction scheme for actin denaturation. A free energy of -28.7(+/-0.2) kJ mol(-1) was determined for the loss of ATP from Ca-free G-actin, in good agreement with previous studies. Understanding the K(eq) value for this step then allowed the temperature dependence of the unfolding reaction of co-factor-free actin to be evaluated, yielding an activation energy for the unfolding of G-actin of 81.3(+/-3.3) kJ mol(-1). By chemical coupling of the extrinsic probe, Alexa Fluor 488 to cysteine 374 of native alpha-actin, we were able to follow the binding and folding of I(3) by CCT, observing for the first time, in vitro re-folding of EDTA-denatured G-actin. The high value of the activation energy between native actin and a non-native folding intermediate (I(3)) is characteristic of a partially folded, molten globule state expected to contain partial secondary structure.
Altschuler
Gabriel M
GM
Cancer Research UK, Centre for Cell and Molecular Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK.
Klug
David R
DR
Willison
Keith R
KR
D000199:Actins / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D018833:Chaperonins / Q000737:chemistry* / Q000378:metabolism; D002614:Chelating Agents / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008958:Models, Molecular; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D011817:Rabbits; D013696:Temperature
D000199:Actins; D002614:Chelating Agents; D005456:Fluorescent Dyes; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D018833:Chaperonins