Examination of the effect of heating on the secondary structure of avidin and avidin-biotin complex by resolution-enhanced two-dimensional infrared correlation spectroscopy

The Fourier transform infrared (FT-IR) spectra of avidin dissolved in D2O i n the absence and the presence of biotin were recorded as a function of tem perature over the range of 25-95 degrees C. At ambient temperature, the spe ctra revealed differences in secondary structure between uncomplexed avidin and avidin complexed to biotin. In the presence of biotin, avidin underwen t slower H-D exchange, demonstrating that when complexed to biotin, avidin adopts a more compact structure that is less accessible to solvent molecule s. In addition, examination of the amide I' band in the spectrum of the avi din-biotin complex revealed the presence of a new intramolecular beta-sheet structure that is less exposed to solvent. Upon heating to 85 degrees C, a ggregation of avidin occurred with the formation of extended intermolecular antiparallel beta-sheet structures. When avidin was complexed to biotin, s ignificant changes in its secondary structure were observed with increasing temperature, but the majority of these changes were found to be reversible upon decreasing the temperature, and no spectral bands associated with agg regate formation were observed over the temperature range examined. The eff ect of increasing temperature on the secondary structure of avidin in the p resence and absence of biotin was further investigated by two-dimensional i nfrared (2D-IR) correlation spectroscopy. The 2D analysis of the spectra of the avidin-biotin complex was facilitated by the use of Fourier self-decon volved spectra to generate the synchronous and asynchronons 2D contour maps , providing very sharp and well-resolved cross peaks. Examination of the sy nchronous and asynchronous contour maps generated from the IR spectra of av idin recorded as a function of increasing temperature revealed that the unf olding of lu-helical structures and the disruption of turns preceded the un folding of the beta-sheet structures within the avidin. Subsequently, the u nfolded protein formed intermolecular antiparallel beta-sheet structures. T he resultant beta-sheet structures did not change upon cooling of the solut ion to ambient temperature. The avidin-biotin complex unfolded via a differ ent pathway, following a sequence of events where the extended beta-sheet s tructure unfolds first, followed by the unfolding of alpha-helical structur es. This behavior in turn is followed by an increase in the antiparallel be ta-sheet structures. Upon cooling of the solution, the avidin-biotin comple x refolded via the same pathway. These studies reveal the potential benefit of employing resolution-enhanced two-dimensional (RE-2D) IR correlation sp ectroscopy in the study of protein dynamics.