Pramodh Vallurupalli

Solution structure of a minor and transiently formed state of a T4 lysozyme mutant

Proteins are inherently plastic molecules, whose function often critically depends on excursions between different molecular conformations (conformers). However, a rigorous understanding of the relation between a protein’s structure, dynamics and function remains elusive. This is because many of the conformers on its energy landscape are only transiently formed and marginally populated (less than a few per cent of the total number of molecules), so that they cannot be individually characterized by most biophysical tools. Here we study a lysozyme mutant from phage T4 that binds hydrophobic molecules and populates an excited state transiently (about 1 ms) to about 3% at 25 °C (ref. 5). We show that such binding occurs only via the ground state, and present the atomic-level model of the ‘invisible’, excited state obtained using a combined strategy of relaxation-dispersion NMR (ref. 6) and CS-Rosetta model building that rationalizes this observation. The model was tested using structure-based design calculations identifying point mutants predicted to stabilize the excited state relative to the ground state. In this way a pair of mutations were introduced, inverting the relative populations of the ground and excited states and altering function. Our results suggest a mechanism for the evolution of a protein’s function by changing the delicate balance between the states on its energy landscape. More generally, they show that our approach can generate and validate models of excited protein states.

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

Molecular function is often predicated on excursions between ground states and higher energy conformers that can play important roles in ligand binding, molecular recognition, enzyme catalysis, and protein folding. The tools of structural biology enable a detailed characterization of ground state structure and dynamics; however, studies of excited state conformations are more difficult because they are of low population and may exist only transiently. Here we describe an approach based on relaxation dispersion NMR spectroscopy in which structures of invisible, excited states are obtained from chemical shifts and residual anisotropic magnetic interactions. To establish the utility of the approach, we studied an exchanging protein (Abp1p SH3 domain)–ligand (Ark1p peptide) system, in which the peptide is added in only small amounts so that the ligand-bound form is invisible. From a collection of 15N, 1HN, 13Cα, and 13CO chemical shifts, along with 1HN-15N, 1Hα-13Cα, and 1HN-13CO residual dipolar couplings and 13CO residual chemical shift anisotropies, all pertaining to the invisible, bound conformer, the structure of the bound state is determined. The structure so obtained is cross-validated by comparison with 1HN-15N residual dipolar couplings recorded in a second alignment medium. The methodology described opens up the possibility for detailed structural studies of invisible protein conformers at a level of detail that has heretofore been restricted to applications involving visible ground states of proteins.

Probing chemical shifts of invisible states of proteins with relaxation dispersion NMR spectroscopy: how well can we do?

Carr-Purcell-Meiboom-Gill relaxation dispersion NMR spectroscopy has evolved into a powerful approach for the study of low populated, invisible conformations of biological molecules. One of the powerful features of the experiment is that chemical shift differences between the exchanging conformers can be obtained, providing structural information about invisible excited states. Through the development of new labeling approaches and NMR experiments it is now possible to measure backbone 13C(alpha) and 13CO relaxation dispersion profiles in proteins without complications from 13C-13C couplings. Such measurements are presented here, along with those that probe exchange using 15N and 1HN nuclei. A key experimental design has been the choice of an exchanging system where excited-state chemical shifts were known from independent measurement. Thus it is possible to evaluate quantitatively the accuracy of chemical shift differences obtained in dispersion experiments and to establish that in general very accurate values can be obtained. The experimental work is supplemented by computations that suggest that similarly accurate shifts can be measured in many cases for systems with exchange rates and populations that fall within the range of those that can be quantified by relaxation dispersion. The accuracy of the extracted chemical shifts opens up the possibility of obtaining quantitative structural information of invisible states of the sort that is now available from chemical shifts recorded on ground states of proteins.

Using relaxation dispersion NMR spectroscopy to determine structures of excited, invisible protein states.

Currently the main focus of structural biology is the determination of static three-dimensional representations of biomolecules that for the most part correspond to low energy (ground state) conformations. However, it is becoming increasingly well recognized that higher energy structures often play important roles in function as well. Because these conformers are populated to only low levels and are often only transiently formed their study is not amenable to many of the tools of structural biology. In this perspective we discuss the role of CPMG-based relaxation dispersion NMR spectroscopy in characterizing these low populated, invisible states. It is shown that robust methods for measuring both backbone chemical shifts and residual anisotropic interactions in the excited state are in place and that these data provide valuable restraints for structural studies of invisible conformers.

Accurate Measurement of Alpha Proton Chemical Shifts of Excited Protein States by Relaxation Dispersion NMR Spectroscopy

Carr-Purcell-Meiboom-Gill relaxation dispersion NMR spectroscopy can provide detailed information about low populated, invisible states of protein molecules, including backbone chemical shifts of the invisible conformer and bond vector orientations that can be used as structural constraints. Notably, the measurement of 1Halpha chemical shifts in excited protein states has not been possible to date because, in the absence of suitable labeling, the homonuclear proton scalar coupling network in side chains of proteins leads to a significant degradation in the performance of proton-based relaxation dispersion experiments. Here we have overcome this problem through a labeling scheme in which proteins are prepared with U-2H glucose and 50% D2O/50% H2O that results in deuteration levels of between 50-88% at the Cbeta carbon. Effects from residual 1Halpha-1Hbeta scalar couplings can be suppressed through a new NMR experiment that is presented here. The utility of the methodology is demonstrated on a ligand binding exchanging system and it is shown that 1Halpha chemical shifts extracted from dispersion profiles are, on average, accurate to 0.03 ppm, an order of magnitude better than they can be predicted from structure using a database approach. The ability to measure 1Halpha chemical shifts of invisible conformers is particularly important because such shifts are sensitive to both secondary and tertiary structure. Thus, the methodology presented is a valuable addition to a growing list of experiments for characterizing excited protein states that are difficult to study using the traditional techniques of structural biology.

Probing structure in invisible protein states with anisotropic NMR chemical shifts.

A general method for obtaining quantitative structural information on invisible, excited protein states by solution-based NMR spectroscopy is presented. The approach exploits relaxation dispersion techniques in which changes in chemical shifts between ground and excited states are monitored in solutions with and without small amounts of residual molecular alignment. This allows the calculation of differences in chemical shifts induced by alignment that can be directly related to molecular structure, in cases where the orientation and magnitude of the chemical-shift tensor are well defined. An example using carbonyl chemical shifts as probes of a protein-ligand binding reaction is presented to illustrate and validate the method.

Isotope labeling methods for studies of excited protein states by relaxation dispersion NMR spectroscopy

The utility of nuclear magnetic resonance (NMR) spectroscopy as a tool for the study of biomolecular structure and dynamics has benefited from the development of facile labeling methods that incorporate NMR active probes at key positions in the molecule. Here we describe a protocol for the labeling of proteins that facilitates their study using a technique that is sensitive to millisecond conformational exchange processes. The samples necessary for an analysis of exchange dynamics are discussed, using the Abp1p SH3 domain from Saccharomyces cerevisiae as an example. For this system, the time frame for production of each sample, including in vitro refolding, is about 80 h. The samples so produced facilitate the measurement of accurate chemical shifts of low populated, invisible conformers that are part of the exchange pathway. The accuracy of the methodology has been established experimentally and the chemical shifts that are obtained provide important restraints in structure calculations of the excited state.

Complementarity of ensemble and single-molecule measures of protein motion: A relaxation dispersion NMR study of an enzyme complex

Single-molecule fluorescence experiments have shown that the conformation of the complex between Escherichia coli general NAD(P)H:flavin oxidoreductase (FRE) and flavin adenine dinucleotide (FAD) fluctuates over a range of timescales between 10−4 and 1 s. Here we use 15N and 13C relaxation dispersion NMR methods to study millisecond-timescale dynamics in the complex. In this time regime, the protein is extremely flexible, with residues that undergo conformational exchange located throughout the molecule. Three distinct regions of dynamics are quantified, with two of them involving residues making contact to the donor (Tyr-35) and acceptor (FAD) sites that participate in the electron transfer reaction monitored in single-molecule experiments. Modulation of the donor–acceptor distance through these conformational exchange processes, occurring with rates of ≈400 and 1,200 s−1 (22°C), affects the rate of electron transfer and partially accounts for the range of the observed dynamics monitored in the fluorescence experiments.

Visualizing Side Chains of Invisible Protein Conformers by Solution NMR

Sparsely populated and transiently formed protein conformers can play key roles in many biochemical processes. Understanding the structure function paradigm requires, therefore, an atomic-resolution description of these rare states. However, they are difficult to study because they cannot be observed using standard biophysical techniques. In the past decade, NMR methods have been developed for structural studies of these elusive conformers, focusing primarily on backbone (1)H, (15)N and (13)C nuclei. Here we extend the methodology to include side chains by developing a (13)C-based chemical exchange saturation transfer experiment for the assignment of side-chain aliphatic (13)C chemical shifts in uniformly (13)C labeled proteins. A pair of applications is provided, involving the folding of β-sheet Fyn SH3 and α-helical FF domains. Over 96% and 89% of the side-chain (13)C chemical shifts for excited states corresponding to the unfolded conformation of the Fyn SH3 domain and a folding intermediate of the FF domain, respectively, have been obtained, providing insight into side-chain packing and dynamics.

Quantifying two-bond 1HN-13CO and one-bond 1H(alpha)-13C(alpha) dipolar couplings of invisible protein states by spin-state selective relaxation dispersion NMR spectroscopy.

Relaxation dispersion NMR spectroscopy has become a valuable probe of millisecond dynamic processes in biomolecules that exchange between a ground (observable) state and one or more excited (invisible) conformers, in part because chemical shifts of the excited state(s) can be obtained that provide insight into the conformations that are sampled. Here we present a pair of experiments that provide additional structural information in the form of residual dipolar couplings of the excited state. The new experiments record (1)H spin-state selective (13)CO and (13)C(alpha) dispersion profiles under conditions of partial alignment in a magnetic field from which two-bond (1)HN-(13)CO and one-bond (1)H(alpha)-(13)C(alpha) residual dipolar couplings of the invisible conformer can be extracted. These new dipolar couplings complement orientational restraints that are provided through measurement of (1)HN-(15)N residual dipolar couplings and changes in (13)CO chemical shifts upon alignment that have been measured previously for the excited-state since the interactions probed here are not collinear with those previously investigated. An application to a protein-ligand binding reaction is presented, and the accuracies of the extracted excited-state dipolar couplings are established. A combination of residual dipolar couplings and chemical shifts as measured by relaxation dispersion will facilitate a quantitative description of excited protein states.