Probing Supra-tc Conformational Exchange in proteins: Insight into Molecular Recognition
Internal motions with diverse timescale (ps–ms) play a crucial role to govern protein function. Molecular recognition events are strongly influenced by motions between the globular rotational correlation time (τc ~ 4 ns) and 40 µs1, known as supra-τc window.2 This previously hidden timescale window came into evidence from RDC-enhanced structural ensembles of Ubiquitin.2 I will present how we have extended the kinetic limit of detection to 2.5 µs by using high power spin-lock in R1ρ relaxation dispersion (RD) experiments, which enabled us to detect the supra-τc motion directly and precisely. These experiments showed that side chains of both ubiquitin and the third immunoglobulin binding domain of protein G (gb3) move on the µs timescale through redistribution of the populations of their side-chain rotamers, which interconvert on the ps to ns timescale, making it likely that this “population shuffling” process is a general mechanism.3
The high spin-lock power R1ρ RD experiments also facilitated the detection of supra-τc motion for the first time in the backbone of GB3 protein, which is routinely being studied by NMR spectroscopists for several years. Backbone dynamics at various temperatures between 262 K and 275 K, in super-cooled conditions, revealed the existence of a global motion in the first β-turn (G9–K13) region of GB3. Lower supra-τc order parameters and enhanced fluctuations in the RDC ensemble also indicate the plasticity of the first β-turn region to one-digit microsecond timescale motion.
Interestingly, the same region of the protein takes part in binding during antibody recognition. Energy landscape, obtained via Eyring relationship between the exchange rate and temperature, indicates that the conformational exchange in GB3 involves multiple conformers within ground state.
1. Michielssens, S.; Peters, J. H.; Ban, D.; Pratihar, S.; Seeliger, D.; Sharma, M.; Giller, K.; Sabo, T. M.;
Becker, S.; Lee, D.; Griesinger, C.; de Groot, B. L., A designed conformational shift to control protein
binding specificity. Angewandte Chemie 2014, 53 (39), 10367-71.
2. Lange, O. F.; Lakomek, N. A.; Fares, C.; Schroder, G. F.; Walter, K. F.; Becker, S.; Meiler,
J.; Grubmuller, H.; Griesinger, C.; de Groot, B. L., Recognition dynamics up to microseconds
revealed from an RDC-derived ubiquitin ensemble in solution. Science 2008, 320 (5882), 1471-5.
3. Smith, C. A.; Ban, D.; Pratihar, S.; Giller, K.; Schwiegk, C.; de Groot, B. L.; Becker, S.; Griesinger, C.;
Lee, D., Population shuffling of protein conformations. Angewandte Chemie 2015, 54 (1), 207-10.