Measurement of bond vector orientations in invisible excited states of proteins

Abstract

The focus of structural biology is on studies of the highly populated, ground states of biological molecules; states that are only sparsely and transiently populated are more difficult to probe because they are invisible to most structural methods. Yet, such states can play critical roles in biochemical processes such as ligand binding, enzyme catalysis, and protein folding. A description of these states in terms of structure and dynamics is, therefore, of great importance. Here, we present a method, based on relaxation dispersion NMR spectroscopy of weakly aligned molecules in a magnetic field, that can provide such a description by direct measurement of backbone amide bond vector orientations in transient, low populated states that are not observable directly. Such information, obtained through the measurement of residual dipolar couplings, has until now been restricted to proteins that produce observable spectra. The methodology is applied and validated in a study of the binding of a target peptide to an SH3 domain from the yeast protein Abp1p and subsequently used in an application to protein folding of a mutational variant of the Fyn SH3 domain where 1 H-15 N dipolar couplings of the invisible unfolded state of the domain are obtained. The approach, which can be used to obtain orientational restraints at other sites in proteins as well, promises to significantly extend the available information necessary for providing a site-specific characterization of structural properties of transient, low populated states that have to this point remained recalcitrant to detailed analysis.olution NMR spectroscopy is a powerful technique for the study of biomolecular dynamics spanning a range of time scales from picoseconds for bond vector librations to many hours for hydrogen exchange in the buried interiors of proteins (1, 2). One very important approach, based on the concept of a ''spin-echo'' that was first described by Hahn in 1950 (3), is called the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion method (4, 5). This class of experiment provides a window into processes with conformational exchange on the millisecond time scale (6), a time regime that is often the relevant one for the lifetimes of bound ligands (7,8), protein folding events (9), or molecular rearrangements that are important for the control of enzyme function (10-13). For systems in which the ground state exchanges with a minor conformer populated at 0.5% or higher and with exchange rates on the order of a hundred to a few thousand per second, the CPMG dispersion experiment provides a sensitive measure of the exchange dynamics (6). Rates of exchange, populations of exchanging states, and chemical shifts of nuclear spins in minor states can be obtained from fits of dispersion profiles to the appropriate model of exchange. Most importantly, information from potentially every residue is obtained in states that are often invisible in even the most sensitive of NMR spectra. Fig. 1a illustrates a simple case in which a loop of a protein, highlighted ...

Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy

“…Relaxation dispersion profiles have been recorded that focus on 15 N, 1 HN, 13 C ␣ , and 13 CO nuclei using previously published experiments (6,(19)(20)(21). Data from a given nucleus-type is fitted together to extract absolute values of chemical shift differences between ground (P) and excited (PL) states, ͉⌬ ͉ (ppm) or ͉⌬ ͉ (rad/sec), along with the global parameters, k ex ϭ k on [L] ϩ k off and p PL , the population of the excited state (3,7). For each of the four nuclei indicated above, the sign of ⌬ can be obtained in many cases and hence the chemical shifts of the excited state, following the approach of Skrynnikov et al (5).…”

“…Thus, with the development of robust methods for the measurement of both chemical shifts and anisotropic interactions in invisible protein states, it is now possible to contemplate structural studies of these ''elusive'' conformers. Here we study the invisible bound state that is formed on the addition of a small mole fraction (Ϸ5%) of a 17-residue Ark1p peptide (17) to a solution of the Abp1p SH3 domain (18), an exchanging system that we have investigated by CPMG relaxation dispersion methods and for which large amounts of chemical shift (6) and RDC/RCSA data have been recorded (7,9,10). We show that restraints available from relaxation dispersion data alone permit the calculation of a well defined ensemble of structures of the invisible bound state, with a pairwise root mean square deviation of backbone atoms of 0.33 Ϯ 0.08 Å. Cross-validation using 1 H- 15 N RDC values measured in a second ''orthogonal'' alignment frame establishes the accuracy of the structure.…”

“…It has also recently been shown that variants of the standard CPMG-based relaxation dispersion experiments can be designed for samples dissolved in small amounts of alignment media (7,8) so that anisotropic interactions, including residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs) of the invisible state, can be measured. Based on these approaches, accurate values of 1 HN-15 N, 1 H ␣ -13 C ␣ , and 1 HN-13 CO RDCs and 13 CO RCSAs have been reported in an exchanging protein system (7,9,10).…”

Electrostatic Interactions in the Binding Pathway of a Transient Protein Complex Studied by NMR and Isothermal Titration Calorimetry

“…The combination of nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (32) can characterize binding kinetics that are too rapid for standard stopped-flow techniques, on the order of 10 4 s Ϫ1 (33). In this approach, a substoichiometric amount of ligand is added to a protein sample whose signals are monitored by NMR spectros- copy.…”

NMR reveals novel mechanisms of protein activity regulation

“…40,53,82,83,87 Incorporation of chemical shifts in NMR structure refinement and the development of new NMR experiments that can report on internuclear vector orientations in excited conformational states provide valuable structural constraints that may enable direct structural characterization of higher energy protein conformations. [88][89][90] Kay and coworkers recently reported the structure of an ''invisible'' protein folding intermediate, a remarkable achievement convincingly showing that structure determination of weakly populated conformational states are within reach. 91 Under favorable conditions, NMRdetected paramagnetic relaxation enhancement (PRE) can be used to determine the structure of weakly populated conformational states.…”