Observation of persistent α‐helical content and discrete types of backbone disorder during a molten globule to ordered peptide transition via deep‐UV resonance Raman spectroscopy

Abstract

The molten globule state can aide in the folding of a protein to a functional structure and is loosely defined as an increase in structural disorder with conservation of the ensemble secondary structure content. Simultaneous observation of persistent secondary structure content with increased disorder has remained experimentally problematic. As a consequence, modeling how the molten globule state remains stable and how it facilitates proper folding remains difficult due to a lack of amenable spectroscopic techniques to characterize this class of partially unfolded proteins. Previously, deep-UV resonance Raman (dUVRR) spectroscopy has proven useful in the resolution of global and local structural fluctuations in the secondary structure of proteins. In this work, dUVRR was employed to study the molten globule to ordered transition of a model four-helix bundle protein, HP7. Both the average ensemble secondary structure and types of local disorder were monitored, without perturbation of the solvent, pH, or temperature. The molten globule to ordered transition is induced by stepwise coordination of two heme molecules. Persistent dUVRR spectral features in the amide III region at 1295–1301 and 1335–1338 cm−1 confirm previous observations that HP7 remains predominantly helical in the molten globule versus the fully ordered state. Additionally, these spectra represent the first demonstration of conserved helical content in a molten globule protein. With successive heme binding significant losses are observed in the spectral intensity of the amide III3 and S regions (1230–1260 and 1390 cm−1, respectively), which are known to be sensitive to local disorder. These observations indicate that there is a decrease in the structural populations able to explore various extended conformations, with successive heme binding events. DUVRR spectra indicate that the first heme coordination between two helical segments diminishes exploration of more elongated backbone structural conformations in the inter-helical regions. A second heme coordination by the remaining two helices further restricts protein motion.

Effects of fluidity on the ensemble structure of a membrane embedded α‐helical peptide

“…CD spectroscopy has many advantages and is often used for rapid assessment of protein structure. However, studies on smaller nonglobular proteins show that the CD and dUVRR spectra can suggest dramatically different secondary structure distributions . Similar discrepancies were observed between the CD and dUVRR spectra of melittin in DMPC liposomes at 37°C and with 5α‐cholestan‐3β‐ol, where a large loss of helical structure could be inferred from the CD spectra (Figures and ), but the dUVRR spectra indicated that the helical structure of melittin remains largely intact.…”

“…The intensity of the amide III band at 1240 cm −1 increases concurrently with a decrease in the negative ellipticity at 222 nm (Figure ). The position and intensity of the lowest frequency amide III band has been shown to be dependent on the ψ dihedral angle . The appearance of a band at 1240 cm −1 is consistent with formation of a small amount of 3 10 ‐helical structure, possibly at the termini of the α‐helix .…”

“…However, an increase in the amide III intensity at 1240 cm −1 can be observed with increasing temperature in the spectra above 22°C. Given that there is little change in the amide S intensity, this indicates that the helical structure of the melittin remains intact …”

Recent advances in linear and nonlinear Raman spectroscopy. Part VIII

“…Brown and coworkers used deep‐UV resonance Raman spectroscopy to observe persistent α‐helical content and discrete types of backbone disorder during a molten globule to ordered peptide transition. Among other results, their observations indicate that there is a decrease in the structural populations able to explore various extended conformations with successive heme binding events . Di et al .…”

Deep ultraviolet resonance Raman spectroscopy as a tool for biomolecule analysis

“…However, an increase in the amide III intensity at 1240 cm -1 can be observed with increasing temperature in the spectra above 22°C. Given that there is little change in the amide S intensity, this indicates that the helical structure of the melittin remains intact 58 .…”