Effects of Protein–pheromone Complexation on Correlated Chemical Shift Modulations

Abstract

Major urinary protein (MUP) is a pheromone-carrying protein of the lipocalin family. Previous studies by isothermal titration calorimetry (ITC) show that the affinity of MUP for the pheromone 2-methoxy-3-isobutylpyrazine (IBMP) is mainly driven by enthalpy, with a small unfavourable entropic contribution. Entropic terms can be attributed in part to changes in internal motions of the protein upon binding. Slow internal motions can lead to correlated or anti-correlated modulations of the isotropic chemical shifts of carbonyl C' and amide N nuclei. Correlated chemical shift modulations (CSM/CSM) in MUP have been determined by measuring differences of the transverse relaxation rates of zero- and double-quantum coherences ZQC{C'N} and DQC{C'N}, and by accounting for the effects of correlated fluctuations of dipole-dipole couplings (DD/DD) and chemical shift anisotropies (CSA/CSA). The latter can be predicted from tensor parameters of C' and N nuclei that have been determined in earlier work. The effects of complexation on slow time-scale protein dynamics can be determined by comparing the temperature dependence of the relaxation rates of APO-MUP (i.e., without ligand) and HOLO-MUP (i.e., with IBMP as a ligand).

Structure, Dynamics and Thermodynamics of the Human Centrin 2/hSfi1 Complex

“…This confirms previous observations on various proteins studied by this method. [38][39][40] For the complex with 1 mM Ca 2+ , all R CSM/CSM rates lie within one standard deviation of the average 〈R CSM/CSM 〉. In contrast, at 4 mM Ca 2+ , contributions that significantly differ from the average were observed for residues L112, D114, D115 and G119 (Ca 2+ -binding site III and its vicinity) and for E156 and S158 (binding site IV; see Fig.…”

“…48 Slow processes on a microsecond-to-millisecond timescale were investigated by measuring multiple-quantum relaxation rates ZQC(C ± N ∓ ) and DQC(C ± N ± ) involving the backbone carbonyl 13 C′ of residue (i − 1) and the amide nitrogen 15 N of residue (i). As shown elsewhere, 36,40 the difference between the ZQC and DQC relaxation rates ΔR = 1/2[R(DQC) − R(ZQC)] is determined by contributions from cross-correlated dipole-dipole (DD/DD) and chemical shift anisotropy (CSA/CSA) effects, as well as isotropic chemical shift modulations (CSM/CSM):…”

Slow backbone dynamics of chicken villin headpiece subdomain probed by NMR C′N cross‐correlated relaxation

“…These empirical rules allow the calculations of the fully anisotropic CSA tensor components (denoted by σ ii ) from the values of isotropic chemical shift based on the specific motional models. In analogy to recent works, [25,26] we have chosen the following tensor components: σ zz C = 83.6 ppm, σ xx C = 251.2 ppm and σ yy C = 3σ iso C − 334.9 ppm for C and σ zz N = 57.7 ppm, σ xx N = σ iso N + 105.5 ppm and σ yy N = 2σ iso N − 163.2 ppm for amide 15 N. Here σ iso stands for the isotropic chemical shift. 15 N chemical shifts were taken from the previously reported assignments, and 13 C chemical shifts assignments were performed according to standard procedures.…”

“…[26,38] A number of residues display CSM/CSM contributions and hence participate in correlated slow motions at 8…”

“…Examples include variations of ϕ and ψ dihedral angles of the backbone, slow collective motions of protein domains, dynamic intramolecular interactions, the existence of transient hydrogen bonds, and transient interactions between complexes or fluctuating interactions with metal ions. [23 -25] The experiment has been recently used to study the microseconds to milliseconds dynamics of the C-terminal domain of human centrin 2 (C-HsCen2) complex with P1-XPC by Kateb et al, [25] major urinary protein in the presence or absence of a pheromone, [26] and dematin headpiece C-terminal domain. [19] In this work we investigate correlated slow motions in neighboring carbonyl and nitrogen nuclei belonging to the same peptide plane and compare the results with the earlier works in order to obtain a global picture of slow backbone dynamics in HP36.…”