Using an amino acid fluorescence resonance energy transfer pair to probe protein unfolding: application to the villin headpiece subdomain and the LysM domain.
Previously, we have shown that p-cyanophenylalanine (Phe CN) and tryptophan (Trp) constitute an efficient fluorescence resonance energy transfer (FRET) pair that has several advantages over commonly used dye pairs. Here, we aim to examine the general applicability of this FRET pair in protein folding-unfolding studies by applying it to the urea-induced unfolding transitions of two small proteins, the villin headpiece subdomain (HP35) and the lysin motif (LysM) domain. Depending on whether Phe CN is exposed to solvent, we are able to extract either qualitative information about the folding pathway, as demonstrated by HP35, which has been suggested to unfold in a stepwise manner, or quantitative thermodynamic and structural information, as demonstrated by LysM, which has been shown to be an ideal two-state folder. Our results show that the unfolding transition of HP35 reported by FRET occurs at a denaturant concentration lower than that measured by circular dichroism (CD) and that the loop linking helix 2 and helix 3 remains compact in the denatured state, which are consistent with the notion that HP35 unfolds in discrete steps and that its unfolded state contains residual structures. On the other hand, our FRET results on the LysM domain allow us to develop a model for extracting structural and thermodynamic parameters about its unfolding, and we find that our results are in agreement with those obtained by other methods. Given the fact that Phe CN is a non-natural amino acid and, thus, amenable to incorporation into peptides and proteins via existing peptide synthesis and protein expression methods, we believe that the FRET method demonstrated here is widely applicable to protein conformational studies, especially to the study of relatively small proteins.
Glasscock
Julie M
JM
Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Zhu
Yongjin
Y
Chowdhury
Pramit
P
Tang
Jia
J
Gai
Feng
F
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002942:Circular Dichroism; D031541:Fluorescence Resonance Energy Transfer; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009088:Mucoproteins / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D016900:Neurofilament Proteins / Q000737:chemistry* / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014508:Urea
D009088:Mucoproteins; D016900:Neurofilament Proteins; D010446:Peptide Fragments; C010393:lysin, gastropoda; C115223:villin headpiece subdomain peptide; D014508:Urea