Kinetics of the Antibody Recognition Site in the Third IgG‐Binding Domain of Protein G

Abstract

Protein dynamics occurring on aw ide range of timescales play ac rucial role in governing protein function. Particularly,m otions between the globular rotational correlation time (t c )a nd 40 ms( supra-t c window), strongly influence molecular recognition. This supra-t c windoww as previously hidden, owingtoalack of experimental methods.Recently,we have developed ah igh-power relaxation dispersion (RD) experiment for measuring kinetics as fast as 4 ms. Fort he first time,t his method, performed under super-cooled conditions, enabled us to detect ag lobal motion in the first b-turn of the thirdI gG-binding domain of protein G( GB3), which was extrapolated to 371 AE 115 ns at 310 K. Furthermore,t he same residues showt he plasticity in the model-free residual dipolar coupling (RDC) order parameters and in an ensemble encoding the supra-t c dynamics.This b-turn is involved in antibody binding,e xhibiting the potential link of the observed supra-t c motion with molecular recognition.

A Small Molecule Causes a Population Shift in the Conformational Landscape of an Intrinsically Disordered Protein

“…Our scheme includes two fast processes and three slow processes that, respectively, exhibit similar kinetic parameters; this type of multi-pathway kinetic scheme has previously been observed for ubiquitin and GB3. 51,54 Furthermore, our scheme is consistent with the observation that mutation of W76 to alanine completely abrogated RD in p27-D2. W76 participates in all of the inter-state transitions; therefore, the W76A mutation eliminates all possible structural transitions and consequently the associated NMR observable RD.…”

“…The global fit yielded E a fast and E a slow values of 49.7 ± 10.5 kJ/mol (attempt frequency: 3.8 × 10 14 ± 1.1 × 10 14 ) and 46.6 ± 4.6 kJ/mol (attempt frequency: 6.0 × 10 12 ± 3.0 × 10 12 ), respectively (Figure A; see the Supporting Information). Please note that over this narrow temperature range the attempt frequencies cannot be known precisely . Measuring RD at temperatures greater than 290 K was not possible because the exchange lifetimes for the fast process (which is characterized by large conformational amplitudes) become too rapid to be detected with the current experimental parameters.…”

“…Please note that over this narrow temperature range the attempt frequencies cannot be known precisely. 51 Measuring RD at temperatures greater than 290 K was not possible because the exchange lifetimes for the fast process (which is characterized by large conformational amplitudes) become too rapid to be detected with the current experimental parameters. The slow process is characterized by small conformational amplitudes (Table S2), which limit detection at elevated temperatures.…”

“…In conclusion, we have demonstrated that a prototypical IDP, p27-D2, populates multiple, rapidly interconverting conformational states (Figures and ). Although ground-state conformational fluctuations of IDPs are thought to exhibit shallow energy wells with low activation energy barriers, we observed that the ground state for p27-D2 exhibits energetic barriers that have the same magnitude as ground-state motional events observed in some folded proteins. , Our experimental findings provide unique insights in terms of the structural and kinetic features of a multistate conformational landscape into how a small molecule (SJ403) binds to an IDP (p27-D2). The NMR RD methods applied here can be used to define the conformational landscapes of a myriad of other IDPs in the future as a basis for understanding their diverse roles in biology and disease and for investigating their possible interactions with small molecules.…”

Günther Laukien Prize 2019: C. Griesinger and G. Bodenhausen / Bessel Research Award: H. Ovaa

“…They study toxic aggregation states of intrinsically disordered proteins related to degenerative diseases and small molecules that interfere with these toxic aggregation states. Griesinger was an author of a Communication in Angewandte Chemie on the kinetics of the antibody recognition site in an IgG‐binding domain of Protein G …”