To obtain insight into the motional features of proteins for enzymatic function, we studied binding reactions between ferredoxin-NADP(+) reductase (FNR) and ferredoxin (Fd) using isothermal titration calorimetry and NMR-based magnetic relaxation and hydrogen/deuterium exchange (HD(ex)). Fd-FNR binding was accompanied by endothermic reactions and driven by the entropy gain. Component-wise analysis of the net entropy change revealed that increases in the conformational entropy of the Fd-FNR complex contributed largely to stabilizing the complex. Intriguingly, analyses of magnetic relaxation and HD(ex) rates with X-ray B factor implied that Fd binding led to both structural stiffening and softening of FNR. Enhanced FNR backbone fluctuations suggest favorable contributions to the net conformational entropy. Fd-bound FNR further showed that relatively large-scale motions of the C terminus, a gatekeeper for interactions of NADP(+) (H), were quenched in the closed form, thereby facilitating exit of NADP(+) (H). This can provide a first dynamic structure-based explanation for the negative cooperativity between Fd and NADP(+) (H) via FNR.
[Show abstract][Hide abstract] ABSTRACT: L-PGDS [lipocalin-type PG (prostaglandin) D synthase] is a multi-functional protein, acting as a PGD2-producing enzyme and a lipid-transporter. In the present study, we focus on the function of L-PGDS as an extracellular transporter for small lipophilic molecules. We characterize the binding mechanism of human L-PGDS for the molecules, especially binding affinity stoichiometry and driving force, using tryptophan fluorescence quenching, ICD (induced circular dichroism) and ITC (isothermal titration calorimetry). The tryptophan fluorescence quenching measurements revealed that haem metabolites such as haemin, biliverdin and bilirubin bind to L-PGDS with significantly higher affinities than the other small lipophilic ligands examined, showing dissociation constant (K(d)) values from 17.0 to 20.9 nM. We focused particularly on the extra-specificities of haem metabolites and L-PGDS. The ITC and ICD data revealed that two molecules of the haem metabolites bind to L-PGDS with high and low affinities, showing K(d) values from 2.8 to 18.1 nM and from 0.209 to 1.63 μM respectively. The thermodynamic parameters for the interactions revealed that the contributions of enthalpy and entropy change were considerably different for each haem metabolite even when the Gibbs energy change was the same. Thus we believe that the binding energy of haem metabolites to L-PGDS is optimized by balancing enthalpy and entropy change.
[Show abstract][Hide abstract] ABSTRACT: We study the contribution of linear, pairwise atom-positional correlations (covariances) to absolute and relative conformational entropy as calculated by quasi-harmonic analysis of molecular dynamics (MD) trajectories (S-QH and Delta S-QH). By analyzing a total of 25 mu s of MD simulations of ubiquitin and six of its binding partners in bound and unbound states, and 2.4 mu s of simulations of eight different proteins in phosphorylated and unphosphorylated states, we show that Delta S-QH represents a remarkably constant fraction of a quasi-harmonic entropy change obtained if one ignores the contribution of covariance terms and uses mass-weighted atom-positional variances only (Delta S-VAR). In other words, the relative contribution of linear correlations to conformational entropy change for different proteins and in different biomolecular processes appears to be largely constant. Based on this, we establish an empirical relationship between relative quasi-harmonic conformational entropy and changes in crystallographic B-factors induced by different processes, and we use it to estimate conformational-entropic contribution to the free energy of binding for a large set of protein complexes based on their X-ray structures. Our results suggest a simple way for relating other types of dynamical observables with conformational entropy in the absence of information on correlated motions, such as in the case of NMR order parameters.
Journal of Chemical Theory and Computation 06/2012; 8(10-10):3820-3829. DOI:10.1021/ct300082q · 5.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A deuterated protein sample is required for nuclear magnetic resonance (NMR) measurements of a large protein because severe signal broadenings occur because of the high molecular weight. The deuterated sample expressed in (2)H(2)O should subsequently be subjected to a back hydrogen exchange at amide groups. To perform the back exchange, the protein molecule is unfolded or destabilized so that internal residues become accessible to the solvent. However, the refolding yield from the destabilized or unfolded state of a large protein is usually low, leading to a dilemma in NMR measurements of large proteins. In our previous paper [Suzuki, M., et al. (2011) Biochemistry50, 10390-10398], we suggested that an acid-denatured microbial transglutaminase (MTG) consisting of 331 amino acid residues can be recovered effectively under low-salt conditions, escaping from the aggregation-prone intermediate. Here, we demonstrate that proMTG, the pro form of MTG consisting of 376 amino acid residues, can be refolded perfectly from the acid-unfolded state under low-salt conditions, as confirmed by circular dichroism and NMR spectroscopies. By performing the same procedure with a deuterated proMTG expressed in (2)H(2)O, we observed complete back exchanges for internal residues by NMR spectroscopy. Our procedure has potential applications to the back hydrogen exchange of large proteins for NMR measurements.
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