NMR Methods to Study Dynamic Allostery

Abstract

Nuclear magnetic resonance (NMR) spectroscopy provides a unique toolbox of experimental probes for studying dynamic processes on a wide range of timescales, ranging from picoseconds to milliseconds and beyond. Along with NMR hardware developments, recent methodological advancements have enabled the characterization of allosteric proteins at unprecedented detail, revealing intriguing aspects of allosteric mechanisms and increasing the proportion of the conformational ensemble that can be observed by experiment. Here, we present an overview of NMR spectroscopic methods for characterizing equilibrium fluctuations in free and bound states of allosteric proteins that have been most influential in the field. By combining NMR experimental approaches with molecular simulations, atomistic-level descriptions of the mechanisms by which allosteric phenomena take place are now within reach.

Fig 1. Three-dimensional structures of allosteric proteins.

(A) The homodimeric catabolite activator protein (CAP) bound to two molecules of cAMP (green spheres; Protein Data Bank [PDB] identifier 1G6N) [11]. (B) The KIX domain of CREB-binding protein (CBP; blue) in complex with the peptides mixed-lineage leukemia (MLL; top, dark green, residues 2,840−2,858) and phosphorylated kinase-inducible domain (pKID; light green, residues 116−149; PDB identifier 2LXT) [12]. (C) The PBX1 homeodomain (PBX-HD, blue) bound to DNA (green) and the HoxB1 homeodomain peptide (light blue, residues 177−185; PDB identifier 1B72) [13]. (D) The 20S core particle proteasome (20S CP); α- and β-subunits are shown in light and dark blue, respectively (PDB identifier 3C91) [14]. (E) The heterodimeric enzyme imidazole glycerol phosphate synthase (IGPS), subunits HisH (light blue) and HisF (dark blue). The allosteric effector PRFAR (dark green spheres) and the substrate glutamine (light green spheres) are shown (PDB identifier 1OX5) [15]. Prepared using PyMOL (The PyMOL Molecular Graphics System, Version 1.41, Schrödinger LLC).

Fig 2. Relaxation dispersion experiments.

(A) Transitions (exchange) between two states, A and B, causes line broadening of resonances in NMR spectra if the chemical shifts of the two states are different (Δω ≠ 0) and the exchange rate constant, k_ex, is in the micro- to millisecond time range. (B) In the typical experimental setup for CPMG relaxation dispersion measurements, resonance intensities at multiple protein sites (e.g., all backbone amide NH groups) are measured at variable CPMG frequencies (bottom). Relaxation dispersion profiles are obtained by converting these intensities to transverse relaxation rates (top). (C) Analysis of RD profiles yields information on kinetic (k_ex), thermodynamic (fractional populations p_A, p_B), and structural (Δω) parameters of the underlying dynamic exchange process(es). RD experiments provide this information only for protein sites with different local structures in states A and B (Δω ≠ 0).

Fig 3. Magnetization exchange experiments.

(A) In cases in which separate resonances are observed for states A and B, transitions between these states occurring in approximately hundreds of milliseconds can be monitored by magnetization exchange. In these experiments, exchange cross-peaks (shown in red) are observed that are directly related to the interconversion between A and B. (B) Analysis of peak intensities in magnetization exchange spectra with variable delay periods yields kinetic (k_ex) and thermodynamic (p_A, p_B) information at multiple sites (e.g., NH groups) in proteins.

Fig 4. Determination of NMR order parameters.

(A) Processes in the pico- to nanosecond time regime can be probed by experiments that monitor the relaxation rates of different spin modes. Relaxation rates at multiple sites in a protein are determined from exponential fits of resonance intensities in a time series. (B) Analysis of the experimental data within the model-free approach separates nanosecond timescale contributions arising from rotational diffusion of the protein as a whole (τ_c) from (typically) picosecond contributions due to internal bond vector fluctuations, for which amplitudes (S²), timescale (τ_e), and, if applicable, information on additional motions are obtained.

Fig 5. Dynamics from residual dipolar couplings (RDCs).

(A) In isotropic solution, rotational diffusion averages dipolar couplings to zero and only scalar couplings J are observed. Weak molecular alignment of proteins impedes averaging of dipolar couplings to zero, and RDCs greater than or less than zero add to line splittings. (B) Residual dipolar couplings contain site-specific information on the orientation of internuclear vectors with respect to a molecular reference frame. Population-weighted averaged RDCs are observed if internal dynamics cause dipolar vectors to reorient. By combining experimental data from multiple molecular alignment media, structural and dynamic contributions can be separated to extract RDC-derived order parameters.