title: Folding of a Zinc-Finger ββα-Motif Investigated Using Two-Dimensional and Time-Resolved Vibrational Spectroscopy.
abstract: Small proteins provide good model systems for studying the fundamental forces that control protein folding. Here, we investigate the folding dynamics of the 28-residue zinc-finger mutant FSD-1, which is designed to form a metal-independent folded ββα-motif, and which provides a testing ground for proteins containing a mixed α/β fold. Although the folding of FSD-1 has been actively studied, the folding mechanism remains largely unclear. In particular, it is unclear in what stage of folding the α-helix is formed. To address this issue we investigate the folding mechanism of FSD-1 using a combination of temperature-dependent UV circular dichroism (UV-CD), Fourier transform infrared (FTIR) spectroscopy, two-dimensional infrared (2D-IR) spectroscopy, and temperature-jump (T-jump) transient-IR spectroscopy. Our UV-CD and FTIR data show different thermal melting transitions, indicating multistate folding behavior. Temperature-dependent 2D-IR spectra indicate that the α-helix is the most stable structural element of FSD-1. To investigate the folding/unfolding re-equilibration dynamics of FSD-1, the conformational changes induced by a nanosecond T-jump are probed with transient-IR and transient dispersed-pump-probe (DPP) IR spectroscopy. We observe biexponential T-jump relaxation kinetics (with time constants of 80 ± 13 ns and 1300 ± 100 ns at 322 K), confirming that the folding involves an intermediate state. The IR and dispersed-pump-probe IR spectra associated with the two kinetic components suggest that the folding of FSD-1 involves early formation of the α-helix, followed by the formation of the β-hairpin and hydrophobic contacts.
authors.affiliation: Van 't Hoff Institute for Molecular Sciences, University of Amsterdam , Science Park 904, 1098 XH Amsterdam, The Netherlands.
authors.forename: Heleen
authors.initials: H
authors.lastname: Meuzelaar
authors.affiliation: Van 't Hoff Institute for Molecular Sciences, University of Amsterdam , Science Park 904, 1098 XH Amsterdam, The Netherlands.
authors.forename: Matthijs R
authors.initials: MR
authors.lastname: Panman
authors.affiliation: Van 't Hoff Institute for Molecular Sciences, University of Amsterdam , Science Park 904, 1098 XH Amsterdam, The Netherlands.
authors.forename: Chris N
authors.initials: CN
authors.lastname: van Dijk
authors.affiliation: Van 't Hoff Institute for Molecular Sciences, University of Amsterdam , Science Park 904, 1098 XH Amsterdam, The Netherlands.
authors.forename: Sander
authors.initials: S
authors.lastname: Woutersen
mesh_terms: D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D017510:Protein Folding; D013055:Spectrophotometry, Infrared; D013696:Temperature; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics; D016335:Zinc Fingers
chemical_list: D004268:DNA-Binding Proteins; C108466:FSD-1 protein, synthetic; D014157:Transcription Factors