American Chemical Society
bi200410c_si_001.pdf (1.09 MB)

Conformational Flexibility of a Human Immunoglobulin Light Chain Variable Domain by Relaxation Dispersion Nuclear Magnetic Resonance Spectroscopy: Implications for Protein Misfolding and Amyloid Assembly

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journal contribution
posted on 2011-07-05, 00:00 authored by Sujoy Mukherjee, Simon P. Pondaven, Christopher P. Jaroniec
The conformational flexibility of a human immunoglobulin κIV light-chain variable domain, LEN, which can undergo conversion to amyloid under destabilizing conditions, was investigated at physiological and acidic pH on a residue-specific basis by multidimensional solution-state nuclear magnetic resonance (NMR) methods. Measurements of backbone chemical shifts and amide 15N longitudinal and transverse spin relaxation rates and steady-state nuclear Overhauser enhancements indicate that, on the whole, LEN retains its native three-dimensional fold and dimeric state at pH 2 and that the protein backbone exhibits limited fast motions on the picosecond to nanosecond time scale. On the other hand, 15N Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion NMR data show that LEN experiences considerable slower, millisecond time scale dynamics, confined primarily to three contiguous segments of about 5–20 residues and encompassing the N-terminal β-strand and complementarity determining loop regions 2 and 3 in the vicinity of the dimer interface. Quantitative analysis of the CPMG relaxation dispersion data reveals that at physiological pH these slow backbone motions are associated with relatively low excited-state protein conformer populations, in the ∼2–4% range. Upon acidification, the minor conformer populations increase significantly, to ∼10–15%, with most residues involved in stabilizing interactions across the dimer interface displaying increased flexibility. These findings provide molecular-level insights about partial protein unfolding at low pH and point to the LEN dimer dissociation, initiated by increased conformational flexibility in several well-defined regions, as being one of the important early events leading to amyloid assembly.