title: Probing the exposure of tyrosine and tryptophan residues in partially folded proteins and folding intermediates by CIDNP pulse-labeling.
abstract: A nuclear magnetic resonance (NMR) technique has been devised to probe the structures of disordered, partially folded states of proteins at the level of individual amino acid residues. Chemically induced dynamic nuclear polarization (CIDNP) is first generated in exposed aromatic side-chains of the denatured state and then transferred to the high-resolution NMR spectrum of the native state by stimulating rapid refolding of the protein. Crucial improvements in sensitivity were achieved by carrying out the polarization-producing photochemistry in a deoxygenated sample of the disordered state of the protein in a magnetic field of 4.0 T and recording the (1)H NMR spectrum of the refolded native state at 9.4 T (400 MHz). Application of this method to the low pH molten-globule state of alpha-lactalbumin reveals remarkably nativelike environments for the aromatic residues in the primary hydrophobic core of the protein. This result provides compelling evidence that the detailed fold of a protein can be established prior to the formation of the cooperative close-packed native structure.
authors.affiliation: Oxford Centre for Molecular Sciences and the Physical and Theoretical Chemistry Laboratory, Oxford University, United Kingdom.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Lyon
authors.forename: Eui-Sik
authors.initials: ES
authors.lastname: Suh
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Dobson
authors.forename: P J
authors.initials: PJ
authors.lastname: Hore
mesh_terms: D007768:Lactalbumin / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010777:Photochemistry; D011489:Protein Denaturation; D017510:Protein Folding; D014364:Tryptophan / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D014443:Tyrosine; D014364:Tryptophan; D007768:Lactalbumin; D009113:Muramidase