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.     

Measurement of carbonyl chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy: comparison between uniformly and selectively 13C labeled samples

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful method for quantifying chemical shifts of excited protein states. For many applications of the technique that involve the measurement of relaxation rates of carbon magnetization it is necessary to prepare samples with isolated (13)C spins so that experiments do not suffer from magnetization transfer between coupled carbon spins that would otherwise occur during the CPMG pulse train. In the case of (13)CO experiments however the large separation between (13)CO and (13)C(alpha) chemical shifts offers hope that robust (13)CO dispersion profiles can be recorded on uniformly (13)C labeled samples, leading to the extraction of accurate (13)CO chemical shifts of the invisible, excited state. Here we compare such chemical shifts recorded on samples that are selectively labeled, prepared using [1-(13)C]-pyruvate and NaH(13)CO(3,) or uniformly labeled, generated from (13)C-glucose. Very similar (13)CO chemical shifts are obtained from analysis of CPMG experiments recorded on both samples, and comparison with chemical shifts measured using a second approach establishes that the shifts measured from relaxation dispersion are very accurate.     

Probing excited states and activation energy for the integral membrane protein phospholamban by NMR CPMG relaxation dispersion experiments

Phospholamban (PLN) is a dynamic single-pass membrane protein that inhibits the flow of Ca²⁺ ions into the sarcoplasmic reticulum (SR) of heart muscle by directly binding to and inhibiting the SR Ca²⁺ATPase (SERCA). The PLN monomer is the functionally active form that exists in equilibrium between ordered (T state) and disordered (R state) states. While the T state has been fully characterized using a hybrid solution/solid-state NMR approach, the R state structure has not been fully portrayed. It has, however, been detected by both NMR and EPR experiments in detergent micelles and lipid bilayers. In this work, we quantitatively probed the μs to ms dynamics of the PLN excited states by observing the T state in DPC micelles using CPMG relaxation dispersion NMR spectroscopy under functional conditions for SERCA. The ¹⁵N backbone and ¹³C_δ1 Ile-methyl dispersion curves were fit using a two-state equilibrium model, and indicate that residues within domain Ia (residues 1-16), the loop (17-22), and domain Ib (23-30) of PLN undergo μs-ms dynamics (k_ex = 6100 ±800 s^- 1 at 17 °C). We measured k_ex at additional temperatures, which allowed for a calculation of activation energy equal to ∼ 5 kcal/mol. This energy barrier probably does not correspond to the detachment of the amphipathic domain Ia, but rather the energy needed to unwind domain Ib on the membrane surface, likely an important mechanism by which PLN converts between high and low affinity states for its binding partners.     

Probing protein dynamics using temperature jump relaxation spectroscopy

There have been recent advances in initiating and perturbing chemical reactions on very fast timescales, as short as picoseconds, thus making it feasible to study a vast range of chemical kinetics problems that heretofore could not be studied. One such approach is the rapid heating of water solutions using laser excitation. Laser-induced temperature jump relaxation spectroscopy can be used to determine the dynamics of protein motion, an area largely unstudied for want of suitable experimental and theoretical probes, despite the obvious importance of dynamics to protein function. Coupled with suitable spectroscopic probes of structure, relaxation spectroscopy can follow the motion of protein atoms over an enormous time range, from picoseconds to minutes (or longer), and with substantial structural specificity.     

Side-chain interactions in the folding pathway of a Fyn SH3 domain mutant studied by relaxation dispersion NMR spectroscopy

A major challenge to the study of protein folding is the fact that intermediate states along the reaction pathway are generally unstable and thus difficult to observe. Recently developed NMR relaxation dispersion experiments present an avenue to accessing such states, providing kinetic, thermodynamic, and structural information for intermediates with small (greater than or equal to approximately 1%) populations at equilibrium. We have employed these techniques to study the three-state folding reaction of the G48M Fyn SH3 domain. Using (13)C-, (1)H-, and (15)N-based methods, we have characterized backbone and side-chain interactions in the folded, unfolded, intermediate, and transition states, thereby mapping the energy landscape of the protein. We find that the intermediate, populated to approximately 1%, contains nativelike structure in a central beta-sheet, and is disordered at the amino and carboxy termini. The intermediate is stabilized by side-chain van der Waals contacts, yet (13)C chemical shifts indicate that methyl-containing residues remain disordered. This state has a partially structured backbone and a collapsed yet mobile hydrophobic core and thus closely resembles a molten globule. Nonpolar side-chain contacts are formed in the unfolded-intermediate transition state; these interactions are disrupted in the intermediate-folded transition state, possibly allowing side chains to rearrange as they adopt the native packing configuration. This work illustrates the power of novel relaxation dispersion experiments in characterizing excited states that are "invisible" in even the most sensitive of NMR experiments.     

Observation of intermediate states of the human prion protein by high pressure NMR spectroscopy

Background  

Prions as causative agents of transmissible spongiform encephalopathies (TSEs) in humans and animals are composed of the infectious isomer, PrP^Sc, of the cellular prion protein, PrP^C. The conversion and thus the propensity of PrP^C to adopt alternative folds leads to the species-specific propagation of the disease. High pressure is a powerful tool to study the physico-chemical properties of proteins as well as the dynamics and structure of folding intermediates.     

Studying excited states of proteins by NMR spectroscopy.

Protein structure is inherently dynamic, with function often predicated on excursions from low to higher energy conformations. For example, X-ray studies of a cavity mutant of T4 lysozyme, L99A, show that the cavity is sterically inaccessible to ligand, yet the protein is able to bind substituted benzenes rapidly. We have used novel relaxation dispersion NMR techniques to kinetically and thermodynamically characterize a transition between a highly populated (97%, 25 degrees C) ground state conformation and an excited state that is 2.0 kcal mol(-1) higher in free energy. A temperature-dependent study of the rates of interconversion between ground and excited states allows the separation of the free energy change into enthalpic (Delta H = 7.1 kcal mol(-1)) and entropic (T Delta S = 5.1 kcal mol(-1), 25 degrees C) components. The residues involved cluster about the cavity, providing evidence that the excited state facilitates ligand entry.     

Conformational differences between various myoglobin ligated states as monitored by 1H NMR spectroscopy

In paramagnetic metmyoglobin, cyanomyoglobin (CNMb), and deoxymyoglobin, His-36 has a high pK (approximately 8), and the NMR titration behavior of the H-2 resonance is perturbed, due to the presence at low pH of a hydrogen bond with Glu-38, which is broken at high pH. The His-36 H-4 resonance shows no shift with pK approximately 8 because of two opposing chemical shift effects but monitors the titration of nearby Glu-36 (pK = 5.6). In diamagnetic derivatives [(carbon monoxy)myoglobin (COMb) and oxymyoglobin (oxyMb)], the titration behavior of His-36 H-2 and H-4 resonances is normalized (pK approximately 6.8). The very slight alkaline Bohr effect in sperm whale myoglobin (Mb) is interpreted in terms of the pK change of His-36 from deoxyMb to oxyMb and compensating pK changes in the opposite direction of other unspecified groups. In sperm whale COMb at 40 degrees C, the distal histidine (His-64) and His-97 have pK values of 5.0 and 5.9. The meso proton resonances remote from these groups do not show a titration shift, but the nearby gamma-meso proton (pK = 5.3) responds to titration of both histidines, and the upfield Val-68 methyl at -2.3 ppm (pK = 4.7) witnesses the titration of nearby His-64. At 20 degrees C, the latter resonance is reduced in size, and a second resonance occurs at -2.8 ppm, which is insensitive to pH and, hence, more remote from His-64. Both resonances arise from two conformations of Val-68 in slow equilibrium. In oxyMb at 20 degrees C, only the latter resonance is observed, presumably because of the steric restrictions imposed by the hydrogen bond between ligand and His-64 in oxyMb, which is not present in COMb. In oxyMb the pK of His-97 (5.6) is similar to that of the meso proton resonances (5.5) and to the pK of other pH-dependent processes, including the very small acid Bohr effect. It is likely that these processes are controlled by the titration of His-97.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elucidating slow binding kinetics of a protein without observable bound resonances by longitudinal relaxation NMR spectroscopy

We developed a new method to elucidate the binding kinetics k_on and k_off, and the dissociation constant K_D (=k_off/k_on), of protein-protein interactions without observable bound resonances of the protein of interest due to high molecular weight in a complex with a large target protein. In our method, k_on and k_off rates are calculated from the analysis of longitudinal relaxation rates of free resonances measured for multiple samples containing different concentration ratios of ¹⁵N-labeled protein and substoichiometric amounts of the target protein. The method is applicable to interactions that cannot be analyzed by relaxation dispersion spectroscopy due to slow interactions on millisecond to second timescale and/or minimal conformational (chemical shift) change upon binding. We applied the method to binding of the B1 domain of protein G (GB1) to immunoglobulin G, and derived the binding kinetics despite the absence of observable bound GB1 resonances.     

Millisecond protein folding studied by NMR spectroscopy

Proteins are involved in virtually every biological process and in order to function, it is necessary for these polypeptide chains to fold into the unique, native conformation. This folding process can take place rapidly. NMR line shape analyses and transverse relaxation measurements allow protein folding studies on a microsecond-to-millisecond time scale. Together with an overview of current achievements within this field, we present millisecond protein folding studies by NMR of the cold shock protein CspB from Bacillus subtilis.     

Amide temperature coefficients in the protein G B1 domain

Temperature coefficients have been measured for backbone amide ¹H and ¹⁵N nuclei in the B1 domain of protein G (GB1), using temperatures in the range 283–313 K, and pH values from 2.0 to 9.0. Many nuclei display pH-dependent coefficients, which were fitted to one or two pK_a values. ¹H coefficients showed the expected behaviour, in that hydrogen-bonded amides have less negative values, but for those amides involved in strong hydrogen bonds in regular secondary structure there is a negative correlation between strength of hydrogen bond and size of temperature coefficient. The best correlation to temperature coefficient is with secondary shift, indicative of a very approximately uniform thermal expansion. The largest pH-dependent changes in coefficient are for amides in loops adjacent to sidechain hydrogen bonds rather than the amides involved directly in hydrogen bonds, indicating that the biggest determinant of the temperature coefficient is temperature-dependent loss of structure, not hydrogen bonding. Amide ¹⁵N coefficients have no clear relationship with structure.     

Histidine side-chain dynamics and protonation monitored by 13C CPMG NMR relaxation dispersion

The use of ¹³C NMR relaxation dispersion experiments to monitor micro-millisecond fluctuations in the protonation states of histidine residues in proteins is investigated. To illustrate the approach, measurements on three specifically ¹³C labeled histidine residues in plastocyanin (PCu) from Anabaena variabilis (A.v.) are presented. Significant Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion is observed for ¹³C^ε1 nuclei in the histidine imidazole rings of A.v. PCu. The chemical shift changes obtained from the CPMG dispersion data are in good agreement with those obtained from the chemical shift titration experiments, and the CPMG derived exchange rates agree with those obtained previously from ¹⁵N backbone relaxation measurements. Compared to measurements of backbone nuclei, ¹³C^ε1 dispersion provides a more direct method to monitor interchanging protonation states or other kinds of conformational changes of histidine side chains or their environment. Advantages and shortcomings of using the ¹³C^ε1 dispersion experiments in combination with chemical shift titration experiments to obtain information on exchange dynamics of the histidine side chains are discussed.     

Probing RNA dynamics via longitudinal exchange and CPMG relaxation dispersion NMR spectroscopy using a sensitive 13C-methyl label

The refolding kinetics of bistable RNA sequences were studied in unperturbed equilibrium via (13)C exchange NMR spectroscopy. For this purpose a straightforward labeling technique was elaborated using a 2'-(13)C-methoxy uridine modification, which was prepared by a two-step synthesis and introduced into RNA using standard protocols. Using (13)C longitudinal exchange NMR spectroscopy the refolding kinetics of a 20 nt bistable RNA were characterized at temperatures between 298 and 310K, yielding the enthalpy and entropy differences between the conformers at equilibrium and the activation energy of the refolding process. The kinetics of a more stable 32 nt bistable RNA could be analyzed by the same approach at elevated temperatures, i.e. at 314 and 316 K. Finally, the dynamics of a multi-stable RNA able to fold into two hairpin- and a pseudo-knotted conformation was studied by (13)C relaxation dispersion NMR spectroscopy.     

Measuring protein self-association using pulsed-field-gradient NMR spectroscopy: Application to myosin light chain 2

Summary At the millimolar concentrations required for structural studies, NMR spectra of the calcium-binding protein myosin light chain 2 (MLC2) showed resonance line widths indicative of extensive self-association. Pulsed-field-gradient (PFG) NMR spectroscopy was used to examine whether MLC2 aggregation could be prevented by the zwitterionic bile salt derivative 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). PFG NMR measurements indicated that CHAPS was capable of preventing MLC2 self-association, but only at concentrations well above the critical micelle concentration of 7.5 mM. CHAPS was most effective at a concentration of 22.5 mM, where the apparent molecular mass of MLC2 correponded to a protein monomer plus seven molecules of bound detergent. The resolution and sensitivity of 2D ¹⁵N-¹H HSQC spectra of MLC2 were markedly improved by the addition of 25 mM CHAPS, consistent with a reduction in aggregation following addition of the detergent. The average amide nitrogen T₂ value for MLC2 increased from 30 ms in the absence of CHAPS to 56 ms in the presence of 25 mM CHAPS. The results of this study lead us to propose that PFG NMR spectroscopy can be used as a facile alternative to conventional techniques such as analytical ultracentrifugation for examining the self-association of biological macromolecules.     

Species-specific differences in the intermediate states of human and Syrian hamster prion protein detected by high pressure NMR spectroscopy

Human (huPrP) and Syrian hamster (ShaPrP) prion proteins have barriers for mutual infectivity, although they fold into almost an identical structure. The pressure responses of huPrP and ShaPrP characterized by high pressure NMR spectroscopy show differences in their excited states, as monitored by pressure-induced chemical shifts and intensity changes of individual residues in the (15)N/(1)H HSQC spectra. Both proteins fluctuate rapidly between two well folded (native) conformations N(1) and N(2) and less frequently between N and the excited states I(1) and I(2) with local disorder that may present structural intermediates on the way to PrP(Sc). These four structural states can be observed in the hamster and human PrP. At ambient pressure, less than 5 molecules of 10,000 are in the intermediate state I(2). From the structural point of view, the different states are mutually different, particularly in positions strategically important for generating species barriers for infection. The results point to the notion that excited state conformers are important for infection and that their structural differences may crucially determine species barriers for infection.     

High protein mobility in skinned rabbit muscle fibres observed by 1H NMR spectroscopy.

1H NMR spectra of skinned rabbit muscle fibers show a group of relatively sharp resonance lines which presumably originate from highly mobile protein domains. Comparison with the spectrum of myosin subfragment 1 suggests that these signals may come at least partly from mobile regions of the myosin head. NMR could possibly be used to characterize the movements of crossbridges in force generation.     

Simple techniques for the quantification of protein secondary structure by 1H NMR spectroscopy

Previous work by Wishart et al. (in press) and others [(1989) J. Magn. Reson. 83, 441-449; (1990) J. Magn. Reson. 90, 165-176] has shown a strong tendency for protein secondary structure to be manifested in 1H NMR chemical shifts. Based on these earlier results, two techniques have been developed for the quantification of secondary structure in proteins. Both methods allow for the rapid and accurate determination of the percent content of helix, coil, and beta-strand based on the integration (or peak enumeration) of selected portions of either 1-D or 2-D 1H NMR spectra. These new and very simple procedures have been found to compare quite favorably to other well established techniques for secondary structure determination such as CD, Raman and IR spectroscopy.