title: Residue-specific real-time NMR diffusion experiments define the association states of proteins during folding.
abstract: Characterizing the association states of proteins during folding is critical for understanding the nature of protein-folding intermediates and protein-folding pathways, protein aggregation, and disease-related aggregation. To study the association states of unfolded, folded, and intermediate species during protein folding, we have introduced a novel residue-specific real-time NMR diffusion experiment. This experiment, a combination of NMR real-time folding experiments and 3D heteronuclear pulsed field gradient NMR diffusion experiments (LED-HSQC), measures hydrodynamic properties, or molecular sizes, of kinetic species directly during the folding process. Application of the residue-specific real-time NMR diffusion experiments to characterize the folding of the collagen triple helix motif shows that this experiment can be used to determine the association states of unfolded, folded, and kinetic intermediates with transient lifetimes simultaneously. The ratio of the apparent translational diffusion coefficients of the unfolded to the folded form of the triple helix is 0.59, which correlates very well with a theoretical ratio for monomer to linear trimer. The apparent diffusion coefficients of the kinetic intermediates formed during triple helix folding indicate the formation of trimer-like associates which is consistent with previously published kinetic and relaxation data. The residue-specific time dependence of apparent diffusion coefficients of monomer and trimer peaks also illustrates the ability to use diffusion data to probe the directionality of triple helix formation. NMR diffusion experiments provide a new strategy for the investigation of protein-folding mechanisms, both to understand the role of kinetic intermediates and to determine the time-dependent aggregation processes in human diseases.
authors.affiliation: Department of Chemistry, Rutgers University, Piscataway, New Jersey 08854, USA.
authors.forename: Alexei V
authors.initials: AV
authors.lastname: Buevich
authors.forename: Jean
authors.initials: J
authors.lastname: Baum
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D004058:Diffusion; D005998:Glycine / Q000737:chemistry; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*
chemical_list: D011506:Proteins; D000409:Alanine; D005998:Glycine