Recognition and binding of a helix-loop-helix peptide to carbonic anhydrase occurs via partly folded intermediate structures
2010 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 98, no 3, 425-433 p.Article in journal (Refereed) Published
We have studied the association of a fluorescently labeled helix–loop–helix peptide scaffold carrying a benzensulfonamide ligand to carbonic anhydrase using steady state and time-resolved fluorescence spectroscopy. The helix–loop–helix peptide, developed for biosensing applications, is labeled with the fluorescent probe dansyl, which serves as a polarity-sensitive reporter of the binding event. Using maximum entropy analysis of the fluorescence lifetime of the dansyl at 1:1 stoichiometry reveals three characteristic fluorescence lifetime groups, which are interpreted as differently interacting peptide–protein structures. We characterize these as mostly bound but unfolded, bound and partly folded, and strongly bound and folded peptide–protein complexes. Furthermore, analysis of the fluorescence anisotropy decay resulted in three different dansyl rotational correlation times, namely 0.18, 1.2, and 23 ns. Using their amplitudes, we can correlate the lifetime groups with the corresponding fluorescence lifetime group. The 23 ns rotational correlation time, which appears with the same amplitude as a 17 ns fluorescence lifetime, shows that the dansyl fluophorophore follows the rotational diffusion of carbonic anhydrase when it is a part of the folded peptide–protein complex. A partly folded and partly hydrated interfacial structure is manifested by a 8 ns dansyl fluorescence lifetime and a 1.2 ns rotational correlation time. This structure, we believe, is similar to a molten-globule-like interfacial structure which allows faster segmental movements and a higher degree of solvent exposure of dansyl. Excitation of dansyl on the helix–loop–helix peptide through Förster energy transfer from one or several tryptophans in the carbonic anhydrase, shows that the helix–loop–helix scaffold binds to a tryptophan-rich domain of the carbonic anhydrase. We conclude that the binding of the peptide to carbonic anhydrase involves a transition from a disordered to ordered structure of the helix–loop–helix scaffold.
Place, publisher, year, edition, pages
2010. Vol. 98, no 3, 425-433 p.
molecular recognition, protein dynamics, time-resolved fluorescence spectroscopy, disordered proteins, disordered to ordered structural transition
IdentifiersURN: urn:nbn:se:uu:diva-109373DOI: 10.1016/j.bpj.2009.10.038ISI: 000274313200010PubMedID: 20141756OAI: oai:DiVA.org:uu-109373DiVA: diva2:272258