Equilibrium refolding transitions driven by trifluoroethanol and by guanidine hydrochloride dilution are similar in GTPase effector domain: implications to sequence-self-association paradigm.
Protein folding transitions starting from a denatured state play crucial roles in deciding the final fate of a protein. A fundamental question in this regard is the role of the amino acid sequence of the protein. In this context, we have investigated here the equilibrium refolding to a partially folded state of the GTPase effector domain (GED) of dynamin driven by addition of increasing amounts of trifluoroethanol (TFE) and compared it with that driven by progressive dilution of the guanidine hydrochloride (Gdn-HCl) denaturant, which has been reported recently [ ( 2008 ) Protein Science 17 , 1319 - 1325 ]. The structural and dynamics changes as the molecule refolds starting from the Gdn-HCl denatured state have been monitored by circular dichroism, fluorescence, and NMR. The molecule remains a monomer in the TFE limiting case, whereas in the Gdn-HCl case, the molecule self-associates as the denaturant is removed. Even so, the two equilibrium transitions seem to have many similarities. The limiting helical contents are similar, and the regions of progressive increase in millisecond time scale motions, suggestive of slow conformational transitions, are largely the same. Though in the guanidine dilution case the partially folded molecules self-associate and there is multimer-monomer equilibrium, the very high concentration ( approximately 6 M) of guanidine prevents self-association in the case of TFE created species. Taken together, the observations under the drastically different solvation conditions suggest that the GED sequence is designed to self-assemble via helices leading to formation of a fully folded megadalton size assembly. The present observations may also have implications for the folding and association mechanism of the protein. These are important from the point of view of dynamin function.
Chugh
Jeetender
J
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai-400005, India.
Sharma
Shilpy
S
Hosur
Ramakrishna V
RV
D002942:Circular Dichroism; D020558:GTP Phosphohydrolases / Q000737:chemistry* / Q000378:metabolism*; D019791:Guanidine / Q000737:chemistry / Q000494:pharmacology*; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects*; D013050:Spectrometry, Fluorescence; D014270:Trifluoroethanol / Q000737:chemistry / Q000494:pharmacology*
D014270:Trifluoroethanol; D020558:GTP Phosphohydrolases; D019791:Guanidine