Use of the novel fluorescent amino acid p-cyanophenylalanine offers a direct probe of hydrophobic core formation during the folding of the N-terminal domain of the ribosomal protein L9 and provides evidence for two-state folding.
Fluorescence-detected stopped flow measurements are the method of choice for studies of protein folding kinetics. However, the methodology suffers from the limitation that the protein of interest either must contain an intrinsic fluorophore or can tolerate its introduction by mutagenesis. Recently, the cyano (nitrile) analogue of phenylalanine has been proposed for use as a fluorescence analogue. Here we take advantage of this new methodology to monitor the formation of the hydrophobic core during the folding of the N-terminal domain of L9 (NTL9). Phenylalanine 5, which is completely buried in the folded state of NTL9, was replaced with p-cyanophenylalanine (p-cyano-Phe). This derivative reports on the formation of the hydrophobic core. The variant adopts the same fold as wild-type NTL9 and is slightly more stable. Refolding and unfolding were monitored using both guanidine HCl and urea jump experiments. In both cases, plots of the natural log of the observed relaxation rate versus denaturant concentration, so-called chevron plots, exhibited the characteristic V shape expected for two-state folding, and no hint of deviation from linearity was observed at low denaturant concentrations. The stability calculated from the measured folding and unfolding rates is in very good agreement with the value obtained from equilibrium measurements as is the m value. The relative compactness of the transition state for folding as defined by the Tanford beta parameter is identical to that of the wild type. The results illustrate the applicability of p-cyano-Phe analogues in protein folding studies and provide further evidence of two-state folding of NTL9.
Aprilakis
Konstantinos N
KN
Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794-3400, USA.
Taskent
Humeyra
H
Raleigh
Daniel P
DP
D002942:Circular Dichroism; D005456:Fluorescent Dyes / Q000737:chemistry*; D008958:Models, Molecular; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry*; D017510:Protein Folding; D012269:Ribosomal Proteins / Q000737:chemistry*; D013816:Thermodynamics
D005456:Fluorescent Dyes; D012269:Ribosomal Proteins; C049650:ribosomal protein L9; D010649:Phenylalanine