title: Stability of gp41 hairpin and helix bundle assembly probed by combined stacking and circular dichroic approaches.
abstract: The envelope glycoprotein gp41 of HIV-1 undergoes structural rearrangement to form a helix hairpin during the virus-mediated fusion. Previous studies to investigate the folding and stability of hairpin did not monitor the end-to-end distance of the molecule. To directly probe the distance change, rhodamine dye was conjugated to the gp41 recombinant near the N- and C-terminal regions to detect the UV absorption and fluorescence intensity changes induced by the chemical denaturant guanidinium chloride (GdmCl). Using the singly- and doubly-labeled constructs allowed us to distinguish between the hairpin formation and protein oligomerization. A biphasic transition of helical structure for the wild type protein was revealed by circular dichroism measurements while unfolding of the hairpin occurred at 6M GdmCl. The relevance of our study to the fusion inhibitor for HIV-1 was borne out by results on the mutants at the positions within the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) regions. A monophasic transition at lower denaturant concentration was detected for the NHR mutant supporting the concept of differential stability of NHR and CHR helical structure. The conclusion that the observed unstacking of doubly-labeled variant arises principally from the intra-molecular dimers was drawn from the unstacking of the protein labeled in the loop. Remarkably, it is deduced that the hairpin is more stable than the CHR helical structure. A model for denaturation of the helix hairpin bundle was proposed from these results. The biological implications of the findings and further applications of the distance-based approach were discussed.
authors: Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan.
authors: Chung-Chieh
authors: CC
authors: Chang
authors: Shu-Fang
authors: SF
authors: Cheng
authors: Chi-Hui
authors: CH
authors: Lin
authors: Steve Shyh-Long
authors: SS
authors: Chen
authors: Ding-Kwo
authors: DK
authors: Chang
mesh_terms: D002942:Circular Dichroism / Q000379:methods*; D015700:HIV Envelope Protein gp41 / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D055503:Protein Multimerization; D017433:Protein Structure, Secondary
chemical_list: D015700:HIV Envelope Protein gp41