Paul Schanda

A Proton-Detected 4D Solid-State NMR Experiment for Protein Structure Determination

Owing to recent advances in instrumentation, methodology and sample-preparation techniques, solid-state NMR spectroscopy is providing unique insights into biological structures at atomic resolution. Three-dimensional structures of proteins and other biological macromolecules can now be determined that are difficult to characterize by X-ray diffraction or solution NMR. Still, the task remains demanding and new methodological developments are needed to make the structure determination more reliable. While it has been demonstrated that assignments are feasible for proteins with over 200 residues, the structure-determination step remains difficult, mainly because spectral overlap introduces ambiguities in restraint assignments during the process of structure generation. These ambiguities can in some cases be resolved, but remain a potential source of errors. Herein, we present an experimental approach that combines the efficient measurement of longrange proton–proton distances in sparsely isotope-labelled samples with proton-detected 3D and 4D correlation spectroscopy. We demonstrate the method in the context of structure determination of the 76-amino-acid protein ubiquitin. Sparsely distributed protons in an otherwise perdeuterated protein yield highly resolved H spectra. The corresponding samples are produced either by expressing perdeuterated protein followed by H back exchange at about 30% of the exchangeable sites or by using suitable precursors to label exclusively a methyl group of alanine, isoleucine, valine or leucine during protein expression. Under fast magic-angle spinning (55 kHz), coherences in such spin systems are sufficiently long-lived to allow not only dipolar, but also scalar-coupling based polarization transfer. H detection increases the sensitivity by a factor of 8 compared to C detection. Similar deuteration schemes have been exploited in solution-state NMR to increase resolution and sensitivity, and to collect precise NOE restraints. An additional advantage arises in the solid phase. In such samples, even the closest H neighbour often corresponds to a long-range contact and dipolar truncation is not an issue, allowing highly-efficient first-order dipolar-recoupling experiments to be used to obtain distance information. Here we use the dipolar recoupling enhanced by amplitude modulation (DREAM) mixing scheme (Figure 1) that provides particularly high polarization-transfer efficiencies due to its adiabatic nature.

Quantitative analysis of protein backbone dynamics in microcrystalline ubiquitin by solid-state NMR spectroscopy.

Characterization of protein dynamics by solid-state NMR spectroscopy requires robust and accurate measurement protocols, which are not yet fully developed. In this study, we investigate the backbone dynamics of microcrystalline ubiquitin using different approaches. A rotational-echo double-resonance type (REDOR-type) methodology allows one to accurately measure (1)H-(15)N order parameters in highly deuterated samples. We show that the systematic errors in the REDOR experiment are as low as 1% or even less, giving access to accurate data for the amplitudes of backbone mobility. Combining such dipolar-coupling-derived order parameters with autocorrelated and cross-correlated (15)N relaxation rates, we are able to quantitate amplitudes and correlation times of backbone dynamics on picosecond and nanosecond time scales in a residue-resolved manner. While the mobility on picosecond time scales appears to have rather uniform amplitude throughout the protein, we unambiguously identify and quantitate nanosecond mobility with order parameters S(2) as low as 0.8 in some regions of the protein, where nanosecond dynamics has also been revealed in solution state. The methodology used here, a combination of accurate dipolar-coupling measurements and different relaxation parameters, yields details about dynamics on different time scales and can be applied to solid protein samples such as amyloid fibrils or membrane proteins.

Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy

Atom-resolved real-time studies of kinetic processes in proteins have been hampered in the past by the lack of experimental techniques that yield sufficient temporal and atomic resolution. Here we present band-selective optimized flip-angle short transient (SOFAST) real-time 2D NMR spectroscopy, a method that allows simultaneous observation of reaction kinetics for a large number of nuclear sites along the polypeptide chain of a protein with an unprecedented time resolution of a few seconds. SOFAST real-time 2D NMR spectroscopy combines fast NMR data acquisition techniques with rapid sample mixing inside the NMR magnet to initiate the kinetic event. We demonstrate the use of SOFAST real-time 2D NMR to monitor the conformational transition of α-lactalbumin from a molten globular to the native state for a large number of amide sites along the polypeptide chain. The kinetic behavior observed for the disappearance of the molten globule and the appearance of the native state is monoexponential and uniform along the polypeptide chain. This observation confirms previous findings that a single transition state ensemble controls folding of α-lactalbumin from the molten globule to the native state. In a second application, the spontaneous unfolding of native ubiquitin under nondenaturing conditions is characterized by amide hydrogen exchange rate constants measured at high pH by using SOFAST real-time 2D NMR. Our data reveal that ubiquitin unfolds in a gradual manner with distinct unfolding regimes.

Direct observation of the dynamic process underlying allosteric signal transmission.

Allosteric regulation is an effective mechanism of control in biological processes. In allosteric proteins a signal originating at one site in the molecule is communicated through the protein structure to trigger a specific response at a remote site. Using NMR relaxation dispersion techniques we directly observe the dynamic process through which the KIX domain of CREB binding protein communicates allosteric information between binding sites. KIX mediates cooperativity between pairs of transcription factors through binding to two distinct interaction surfaces in an allosteric manner. We show that binding the activation domain of the mixed lineage leukemia (MLL) transcription factor to KIX induces a redistribution of the relative populations of KIX conformations toward a high-energy state in which the allosterically activated second binding site is already preformed, consistent with the Monod-Wyman-Changeux (WMC) model of allostery. The structural rearrangement process that links the two conformers and by which allosteric information is communicated occurs with a time constant of 3 ms at 27 degrees C. Our dynamic NMR data reveal that an evolutionarily conserved network of hydrophobic amino acids constitutes the pathway through which information is transmitted.

Fast Two-Dimensional NMR Spectroscopy of High Molecular Weight Protein Assemblies

An optimized NMR experiment that combines the advantages of methyl-TROSY and SOFAST-HMQC has been developed. It allows the recording of high quality methyl (1)H-(13)C correlation spectra of protein assemblies of several hundreds of kDa in a few seconds. The SOFAST-methyl-TROSY-based experiment offers completely new opportunities for the study of structural and dynamic changes occurring in molecular nanomachines while they perform their biological function in vitro.

Longitudinal-relaxation-enhanced NMR experiments for the study of nucleic acids in solution.

Atomic-resolution information on the structure and dynamics of nucleic acids is essential for a better understanding of the mechanistic basis of many cellular processes. NMR spectroscopy is a powerful method for studying the structure and dynamics of nucleic acids; however, solution NMR studies are currently limited to relatively small nucleic acids at high concentrations. Thus, technological and methodological improvements that increase the experimental sensitivity and spectral resolution of NMR spectroscopy are required for studies of larger nucleic acids or protein-nucleic acid complexes. Here we introduce a series of imino-proton-detected NMR experiments that yield an over 2-fold increase in sensitivity compared to conventional pulse schemes. These methods can be applied to the detection of base pair interactions, RNA-ligand titration experiments, measurement of residual dipolar (15)N-(1)H couplings, and direct measurements of conformational transitions. These NMR experiments employ longitudinal spin relaxation enhancement techniques that have proven useful in protein NMR spectroscopy. The performance of these new experiments is demonstrated for a 10 kDa TAR-TAR*(GA) RNA kissing complex and a 26 kDa tRNA.

Native-unlike Long-lived Intermediates along the Folding Pathway of the Amyloidogenic Protein β2-Microglobulin Revealed by Real-time Two-dimensional NMR

β2-microglobulin (β2m), the light chain of class I major histocompatibility complex, is responsible for the dialysis-related amyloidosis and, in patients undergoing long term dialysis, the full-length and chemically unmodified β2m converts into amyloid fibrils. The protein, belonging to the immunoglobulin superfamily, in common to other members of this family, experiences during its folding a long-lived intermediate associated to the trans-to-cis isomerization of Pro-32 that has been addressed as the precursor of the amyloid fibril formation. In this respect, previous studies on the W60G β2m mutant, showing that the lack of Trp-60 prevents fibril formation in mild aggregating condition, prompted us to reinvestigate the refolding kinetics of wild type and W60G β2m at atomic resolution by real-time NMR. The analysis, conducted at ambient temperature by the band selective flip angle short transient real-time two-dimensional NMR techniques and probing the β2m states every 15 s, revealed a more complex folding energy landscape than previously reported for wild type β2m, involving more than a single intermediate species, and shedding new light into the fibrillogenic pathway. Moreover, a significant difference in the kinetic scheme previously characterized by optical spectroscopic methods was discovered for the W60G β2m mutant.

Direct Detection of 3hJNC′ Hydrogen‐Bond Scalar Couplings in Proteins by Solid‐State NMR Spectroscopy

Hydrogen bonds (H-bonds) are important and ubiquitous interactions in chemistry and biology. They are a key element in proteins and nucleic acids for stabilizing the three-dimensional fold and are thus important for the functionality. The presence of an H-bond can be indirectly deduced from the local geometry as obtained from X-ray or NMR methods, and a variety of NMR parameters depend also on hydrogen bonding (e.g. chemical shifts induced by H-bonds or H quadrupolar coupling constants). Direct evidence of hydrogen bonding, however, is provided by the presence of an H-bond-mediated scalar coupling. Experiments that directly measure J couplings across N H···N and N H···O=C bonds in nucleic acids and proteins, respectively, have been introduced for solution-state NMR spectroscopy and have received great interest. Such experiments allow the direct identification of the donor and acceptor side of each Hbond, as well as the determination of the size of the coupling—which is a very sensitive probe of the geometry around an H-bond. Even though solid-state NMR experiments can also use the dipolar interaction to indirectly probe N H···N hydrogen bonding in nucleic acids 8] or N H···O=C bonds in proteins, the intrinsic through-H-bond nature of the scalar coupling is appealing. J-based correlation spectroscopy has been demonstrated for proteins in the solid state and has been successful in measuring J couplings down to a few Hertz, including N H···N H-bond couplings in crystals of organic molecules . The small values for the scalar coupling constants of Hbonds in proteins, with average values in ubiquitin for N H···O=C H-bond scalar coupling constants (JN,C’) of ( 0.38 0.12) Hz (a-helix) to ( 0.65 0.14) Hz (b-sheet), make these experiments challenging in terms of sensitivity. In liquids, difficulties arise particularly for larger molecules, where the coherence decay owing to faster T2 relaxation greatly attenuates the signal during the long time periods required for the polarization transfer mediated by the small J coupling. The situation is even more challenging in solids because of the presence of secular anisotropic interactions, which provide additional mechanisms of transverse dephasing. The strategies employed to minimize this additional dephasing (and thus minimizes T2’) include the use of high magic-angle-spinning (MAS) frequencies and the dilution of the H spin system by extensive deuteration 20] to avoid the need for high-power proton decoupling over extended time periods. Herein, we demonstrate the use of a deuterated and partially backprotonated microcrystalline protein at high MAS frequencies, to directly measure sub-Hertz trans-Hbond scalar coupling constants. Figure 1 shows strips from a “long-range” 3D HNCO correlation experiment optimized for the detection of JNC’ couplings, and were recorded on a microcrystalline sample of H,C,N-labeled ubiquitin, which was protonated at 20 % of the backbone amides and other exchangeable sites. The pulse sequence used to obtain these data is a purely Jbased HNCO experiment, akin to an experiment proposed by Cordier and Grzesiek for measurement of JNC’ in the solution state. The details of the pulse sequence are shown in Figure S1 of the Supporting Information. The key features of this H-detected experiment are long out-and-back N C’ INEPT blocks for a duration of 2T= 66.6 ms to transfer coherence from the H-bond donor N to the acceptor C’, and was carried out in the presence of low-power (3.1 kHz) H decoupling. The evolution resulting from the large JNC’ coupling (approximately 15 Hz = 1/(2T)) vanishes at multiples of 2T. Therefore, the small JNC’ couplings become detectable and lead to coherence transfer over H-bonds. The data in Figure 1 unambiguously show cross-peaks that arise from transfer over N H···O=C hydrogen bonds involving the amides of residues L15, V17, I44, K6, and L50 (the latter two have overlapping cross-peaks) with the carbonyl groups of I3, M1, H68, L67, and L43. The size of the couplings involved is known to be 0.5–0.6 Hz (see below), which is almost an order of magnitude smaller than the smallest couplings measured to date using solid-state NMR spectroscopy. To confirm the results and to investigate the reproducibility of the measurement, we have performed an additional experiment under slightly different conditions and using a sample with a higher degree of protonation on the exchangeable sites (30 % rather than 20%). The 2D long-range H(N)CO data set obtained on this sample is shown in Figure S2 of the Supporting Information. The spectrum reveals cross-peaks arising from H-bonds of residues F4 and E34, in addition to the ones observed in the 3D spectrum. In the 3D data set of Figure 1 these cross-peaks overlapped with residual one-bond cross-peaks and are resolved in the [*] Dr. P. Schanda, M. Huber, Dr. R. Verel, Dr. M. Ernst, Prof. Dr. B. H. Meier Physical Chemistry, ETH Z rich Wolfgang-Pauli-Strasse 10, 8093 Z rich (Switzerland) Fax: (+ 41)44-632-1621 E-mail: pasa@nmr.phys.chem.ethz.ch beme@ethz.ch Homepage: http://www.ssnmr.ethz.ch

Amplitudes and time scales of picosecond-to-microsecond motion in proteins studied by solid-state NMR: a critical evaluation of experimental approaches and application to crystalline ubiquitin.

Solid-state NMR provides insight into protein motion over time scales ranging from picoseconds to seconds. While in solution state the methodology to measure protein dynamics is well established, there is currently no such consensus protocol for measuring dynamics in solids. In this article, we perform a detailed investigation of measurement protocols for fast motions, i.e. motions ranging from picoseconds to a few microseconds, which is the range covered by dipolar coupling and relaxation experiments. We perform a detailed theoretical investigation how dipolar couplings and relaxation data can provide information about amplitudes and time scales of local motion. We show that the measurement of dipolar couplings is crucial for obtaining accurate motional parameters, while systematic errors are found when only relaxation data are used. Based on this realization, we investigate how the REDOR experiment can provide such data in a very accurate manner. We identify that with accurate rf calibration, and explicit consideration of rf field inhomogeneities, one can obtain highly accurate absolute order parameters. We then perform joint model-free analyses of 6 relaxation data sets and dipolar couplings, based on previously existing, as well as new data sets on microcrystalline ubiquitin. We show that nanosecond motion can be detected primarily in loop regions, and compare solid-state data to solution-state relaxation and RDC analyses. The protocols investigated here will serve as a useful basis towards the establishment of a routine protocol for the characterization of ps–μs motions in proteins by solid-state NMR.

Site-resolved measurement of microsecond-to-millisecond conformational-exchange processes in proteins by solid-state NMR spectroscopy.

We demonstrate that conformational exchange processes in proteins on microsecond-to-millisecond time scales can be detected and quantified by solid-state NMR spectroscopy. We show two independent approaches that measure the effect of conformational exchange on transverse relaxation parameters, namely Carr–Purcell–Meiboom–Gill relaxation-dispersion experiments and measurement of differential multiple-quantum coherence decay. Long coherence lifetimes, as required for these experiments, are achieved by the use of highly deuterated samples and fast magic-angle spinning. The usefulness of the approaches is demonstrated by application to microcrystalline ubiquitin. We detect a conformational exchange process in a region of the protein for which dynamics have also been observed in solution. Interestingly, quantitative analysis of the data reveals that the exchange process is more than 1 order of magnitude slower than in solution, and this points to the impact of the crystalline environment on free energy barriers.