Determination of Leu side-chain conformations in excited protein states by NMR relaxation dispersion.
Fits of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion profiles allow extraction of the kinetics and thermodynamics of exchange reactions that interconvert highly populated, ground state and low populated, excited state conformers. Structural information is also available in the form of chemical shift differences between the interconverting protein states. Here we present a very simple method for extracting chi(2) rotamer distributions of Leu side chains in 'invisible' excited protein states based on measurement of their (13)C(delta1)/(13)C(delta2) chemical shifts using methyl CPMG dispersion experiments. The methodology is applied to study the protein folding reaction of the Fyn SH3 domain. A uniform chi(2) rotamer distribution is obtained for Leu residues of the unfolded state, with each Leu occupying the trans and gauche+ conformations in a 2:1 ratio. By contrast, leucines of an 'invisible' Fyn SH3 domain folding intermediate show a much more heterogeneous distribution of chi(2) rotamer populations. The experiment provides an important tool toward the quantitative characterization of both the structural and dynamics properties of states that cannot be studied by other biophysical tools.
Hansen
D Flemming
DF
University of Toronto, Department of Molecular Genetics, 1 King's College Circle, Toronto, M5S 1A8, ON, Canada.
Neudecker
Philipp
P
Vallurupalli
Pramodh
P
Mulder
Frans A A
FA
Kay
Lewis E
LE
D007930:Leucine / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011487:Protein Conformation; D051076:Proto-Oncogene Proteins c-fyn / Q000737:chemistry*; D013237:Stereoisomerism; D018909:src Homology Domains
D051076:Proto-Oncogene Proteins c-fyn; D007930:Leucine