NMR paves the way for atomic level descriptions of sparsely populated, transiently formed biomolecular conformers

Abstract

The importance of dynamics to biomolecular function is becoming increasingly clear. A description of the structure-function relationship must, therefore, include the role of motion, requiring a shift in paradigm from focus on a single static 3D picture to one where a given biomolecule is considered in terms of an ensemble of interconverting conformers, each with potentially diverse activities. In this Perspective, we describe how recent developments in solution NMR spectroscopy facilitate atomic resolution studies of sparsely populated, transiently formed biomolecular conformations that exchange with the native state. Examples of how this methodology is applied to protein folding and misfolding, ligand binding, and molecular recognition are provided as a means of illustrating both the power of the new techniques and the significant roles that conformationally excited protein states play in biology.

Keywords: energy landscape; invisible states; structure–function paradigm; transiently and sparsely populated biomolecular states.

Fig. 1.

NMR methods for studying sparsely populated, transiently formed biomolecular conformers. (A) Schematic 1D energy landscape showing the ground state of a protein in exchange with a thermally accessible excited state. Exchange between G and E is in the microsecond-to-millisecond regime, with p_E << p_G. (B) Schematic (Left) showing a small region from a standard ¹⁵N-¹H Heteronuclear Single Quantum Coherence dataset. The peak derived from state E (dashed black) is not visible in a typical spectrum and is shown here only for clarity. The CEST profile (Right) is obtained by varying the ¹⁵N frequency of a weak radio frequency (B₁) field. Reduction in resonance intensity (I/I₀) of the ground-state peak is seen when irradiation frequencies correspond to resonance positions of G or E. Consequently, Δϖ_GE can be readily obtained from the CEST profile. (C) CPMG RD profiles, R_2,eff vs. ν_CPMG, for different Δϖ_GE values (0 ppm, black; 1.7 ppm, red; 6.8 ppm, green). Values of R_2,eff are calculated from intensities of correlations derived from the ground state as a function of pulsing frequency. Higher pulsing frequencies more effectively refocus the dephasing (excess peak line width) arising from exchange, resulting in narrower peaks and smaller R_2,eff values. CPMG dispersion profiles can be fit to extract Δϖ_GE values and exchange parameters. (D) Cartoon representation of a protein labeled with a paramagnetic spin label (black) that exchanges between a ground-state conformation and a compact excited state. The shaded circle represents the sphere of influence of the spin label. The regions of the protein that are proximal to the unpaired electron, and, hence the pattern of PREs, are clearly different in the two states.

Fig. 2.

Transient intermediates along the FF domain folding pathway. Structures of the N [Protein Data Bank (PDB) ID code 1UZC for wt] and I (PDB ID codes 2KZG and 2L9V for wt and L24A) states of wt (A) and L24A (B) FF domains. The exact values of rate constants and populations differ slightly depending on the isotope-labeling scheme used and sample conditions. Side chains making nonnative hydrophobic contacts in each of the I states are highlighted and colored according to the secondary structure element from which they originate. Y49 flips from inside the core of the protein to the outside during the L24A FF domain I-to-N transition. (C) A model for FF folding depicting both the folding and the dimerization pathways. (A and B are adapted from ref. .)

Fig. 3.

An excited state of the Fyn SH3 domain at the interface between folding and misfolding. (A) Three-dimensional structures of the N (PDB ID code 2LP5) and I (PDB ID code 2L2P) states of the A39V/N53P/V55L Fyn SH3 domain, color-coded according to the surface aggregation propensity score (S_agg) (91). F4, which fills the position occupied by β5 in N, is shown in stick representation (gray). (B) A39V/N53P/V55L Fyn SH3 Δ(57–60), a structural mimic of I as seen from chemical shift correlations (Left Inset), aggregates in a time-dependent fashion leading to a loss in intensity of NMR resonances in ¹H-¹⁵N HSQC datasets with concomitant formation of amyloid fibrils that can be visualized in an electron micrograph (Right Inset). (Adapted from ref. . Reprinted with permission from AAAS.)

Fig. 4.

Probing the structure–function relationship at different points on the energy landscape. (A) Ground-state X-ray structure of L99A T4L (PDB ID code 3DMV), color-coded according to the magnitude of chemical shift differences (Δϖ_RMS) between the ground and excited states (19). The gray mesh delineates the cavity that results from the L99A mutation. (B and C) Comparison of the C-terminal domain of L99A T4L in the ground (PDB ID code 3DMX) (B) and excited (PDB ID code 2LCB) (C) states, highlighting the different orientations of the F and G helices in each of the conformers. The F114 side chain that rotates into the cavity in the excited state and the bound benzene in the ground state are shown using space-filling representations. (D) The ground and excited-state populations can be manipulated by a small number of mutations. G (Left) and E states become comparable in population in the L99A/G113A construct (Center), as seen in ¹³C-¹H correlation spectra. The new set of peaks (red) have chemical shift values that are in excellent agreement with those obtained for state E from fits of the L99A CPMG RD profiles. Populations of the ground and excited states are inverted in L99A/G113A/R119P T4L (Right), as the ground-state peaks (blue) disappear from the NMR spectrum. The affinity of T4L for ligand in each of the constructs is indicated. [Adapted by permission from Macmillan Publishers Ltd: Nature (19), copyright 2011.]

Fig. 5.

Nucleic acid conformational plasticity can modulate function. (A) Sequence of A₆-DNA, which shows significant local conformational exchange at the highlighted A-T (red) and G-C (blue) base pairs. Molecular structures of Watson–Crick (G state) and Hoogsteen (E state) A-T (Upper) and G-C (Lower) base pairs. Purine residues A and G undergo an anti to syn conformational transition from G to E. (B) Secondary structure and hydrogen-bonding partners in G and E states of the HIV1 TAR apical loop. A pair of noncanonical A-C and G-U base pairs is formed in E.

Fig. 6.

Compact transient conformations in the Ca²⁺ sensor signaling protein calmodulin. (A) Structures of CaM-4Ca²⁺ (green) (PDB ID code 1CLL) and CaM-4Ca²⁺ bound to MLCK peptide (blue) (PDB ID code 1CDL). (B) Superposition of the structures in A best fit to the N-terminal (Left) or C-terminal (Right) domains. Structures of 26 other peptide-bound structures of CaM-4Ca²⁺ are best fit in similar ways and shown superimposed as atomic probability density maps in gray. (C) Atomic probability density maps of the compact excited-state conformations of CaM-4Ca²⁺ shown at contour levels ranging from 0.1 (blue) to 0.5 (red) and best fit to the N-terminal (Left) or C-terminal (Right) domains of free CaM-4Ca²⁺ (dark green). The corresponding density maps of the major state, which lacks significant interdomain PREs, are shown in gray at a contour level of 0.1. [Adapted with permission from ref. (Copyright 2011, American Chemical Society).]