Chemistry at Illinois University of Illinois at Urbana-Champaign

Chad M. Rienstra

Professor of Chemistry
Affiliate, Biophysics and Computational Biology
Affiliate, Biochemistry

Professor Rienstra received his B.A. degree in chemistry from Macalester College (St. Paul, MN) in 1993 and his Ph.D. at the Massachusetts Institute of Technology in 1999. He was a postdoctoral fellow at Columbia University and joined the faculty at Illinois in 2002. His research interests are in solid state nuclear magnetic resonance (SSNMR), including the development of new pulse sequence methodology and instrumentation, and application to studies of protein structure and dynamics.

Research

Research in the Rienstra group aims to establish solid state nuclear magnetic resonance (SSNMR) as a preferred method for routine, atomic resolution structural and dynamic analysis of biological macromolecules. The field of SSNMR is in the midst of a revolution not unlike the advances in X-ray crystallography that occurred during the 1970's, and solution NMR in the 1980's. Those methods have been responsible for the vast majority of all protein structures known to date. However, neither method has been applied in a general fashion to membrane proteins and protein-lipid complexes, which have profound importance to biochemistry, yet remain vastly underrepresented in the database of known protein structures. SSNMR has two distinct advantages relevant to the study of biological membranes. First, unlike in solution NMR, where global correlation (tumbling) times impose fundamental restrictions of the particle size that may be studied, in the solid state, spectral intensities and homogeneous line widths of individual NMR signals do not depend upon molecular weight. Second, unlike in crystallography, in our SSNMR experiments long-range order is not required, because the inhomogeneous line widths are determined by the degree of order in the local (5 to 10 Å) environment; therefore heterogeneous sample environments (e.g., asymmetric oligomeric assemblies, protein-lipid complexes, etc.) are inherently no less qualified for structural analysis by SSNMR than single crystals. These two characteristics make SSNMR the best (and often the only) method for studying atomic resolution structure in biological membranes, precipitated peptide aggregates (e.g., β-amyloid peptides), glasses, frozen solutions and lyophilized powders (e.g., trapped enzyme-substrate intermediates). Therefore intense interest has been focused on the problem of global structure determination by SSNMR.

Multi-dimensional SSNMR methods are in a relatively early stage of development, due to a variety of technical limitations that have been overcome in recent years, and a number of ongoing challenges that we intend to address. Historically, solid state NMR studies were performed at low field (200 to 400 MHz), using samples labeled at only a few positions with spin-1/2 (13C, 15N) nuclei, in order to acquire 1D spectra with inherently low resolution. At Illinois, we are upgrading the School of Chemical Sciences 500 MHz wide bore and 750 MHz NMR spectrometers to perform state-of-the-art, high resolution, multi-dimensional, triple and quadruple resonance magic-angle spinning experiments. Only a handful of laboratories worldwide currently have access to such high-field magnets for SSNMR. Furthermore, through both collaborations with major spectrometer vendors and independent efforts in Urbana, we are building several new types of magic-angle spinning SSNMR probes for novel experiments. We intend to leverage these capabilities in combination with 2D, 3D, and 4D correlation methods to resolve all of the hundreds to thousands of NMR signals in proteins that have been uniformly enriched with 15N and 13C isotopes, thereby increasing throughput by 2 to 3 orders of magnitude compared to traditional SSNMR methods. With resolved and assigned signals, a variety of quantum mechanical observables (such as dipolar and quadrupolar couplings, isotropic and anisotropic chemical shifts) can be uniquely measured and mapped to each individual site, to reveal structural and dynamic properties (internuclear distances, torsion angles, and relaxation rates).

Projects in three specific areas are being pursued:

  1. Studies of globular protein structure and dynamics.

    Several groups recently have made progress towards full chemical shift assignments of uniformly-15N,13C-labeled solid proteins (e.g., BPTI, α-spectrin SH3, ubiquitin), and low resolution de novo SSNMR structures of such globular proteins are imminent. However, there is not yet an established suite of SSNMR experiments suitable in the general case for structure determination. We intend to develop new experiments where required, and apply refined versions of experiments currently in the literature, to resolve, assign, and map structural constraints onto all sites in uniformly labeled proteins. All such experiments will be at minimum 3D: two (or three) dimensions of chemical shifts to resolve sites in the protein uniquely, and a third (or fourth) dimension to acquire structural and dynamic data. These efforts at first will primarily be methodological, in order to establish that SSNMR can independently solve and refine structures to similar or better resolution than solution NMR and crystallography (

    As an important aspect of this work, we will perform detailed studies of side-chain conformation and dynamics. In cases where ultra-high resolution crystal structures are available, data increasingly indicate that multiple side-chain rotameric sub-states are populated, even in so-called static structures. Furthermore, preliminary evidence shows that rotameric inter-conversion plays in integral role in ligand binding, release of product from enzyme active sites, and the events associated with ion translocation in membranes. However, crystallography is not well suited to distinguish between static disorder and dynamic processes. Therefore we will develop SSNMR experiments to measure rotamer populations and exchange rates over a range of temperatures (350 K), in order to quantify activation energies and entropic contributions to such events, and to understand the correlation of dynamic events between sites distant in primary protein structure. As in solution NMR, solid state measurements of relaxation parameters can probe the nanosecond to microsecond motional regimes, and chemical exchange line shapes report upon millisecond to second motions. Moreover, in the solid state, measurements of fast (picosecond) librational amplitudes derive directly from the tensor magnitudes inherently to the dipolar spectra (these measurements often have a precision of ±1%, whereas similar order parameter analysis by solution NMR has much poorer precision). Thus solid state NMR can probe motion over 12 to 15 orders of magnitude, in a site-specific manner. We expect that availability of such data will lend insight into the thermostability of globular proteins, and the forces that drive assembly of membrane protein complexes, where specific interactions between side chains are the primary difference between protein-protein and protein-lipid interactions in this hydrophobic context.

  2. Instrumentation and experimental methods for improved spectral sensitivity.

    One major challenge in the solid state is to improve sensitivity in a manner that is generally applicable to many molecular systems of interest. Current state-of-the-art instruments yield sufficient sensitivity to acquire 2D spectra of ~50 kDa effective molecular weight asymmetric units in ~12 hours, and 3D spectra in 24 to 48 hours. An order of magnitude increase in signal-to-noise would permit such experiments to be performed in a matter of minutes, and/or asymmetric units of ~500 kDa to be studied in reasonable time frames. To this end, we are designing and building magic-angle spinning probes, receiver systems, and related hardware specifically for the purpose of proton signal detection with optimal efficiency. The instrumentation is intended for multiple-pulse decoupling experiments at high rates of magic-angle spinning, and optimal receiver recovery and noise figure. The potential gain in sensitivity relative to 13C and 15N signal detection is a factor of 10 to 30, or a factor of 100 to 1,000 in experiment time.

  3. Applications to membrane proteins.

    In collaboration with the Gennis laboratory, we will apply high resolution magic-angle spinning methods to the cytochrome c oxidase enzyme complex. We are especially interested in the conformational changes that occur upon protonation and deprotonation of Asp and Glu side-chain carboxylic acid signals. Changes in isotropic chemical shifts upon protonation for such signals are 4±2 ppm, which is easily detectable in SSNMR microcrystals or precipitates. Therefore pH titrations by SSNMR to determine pKa's in a site-specific manner are quite feasible. Likewise, side-chain conformational exchange of residues nearby Asp and Glu sites is expected, and can be identified by the 5 to 10 ppm chemical shift changes that occur upon side-chain rotameric conversion. It is exactly in the context of such measurements that the greatest strengths of SSNMR are evident, in probing the chemical events of protonation and side-chain conformational exchange. We expect that mechanistic proposals will be validated or dismissed based on such data.

Publications

A comprehensive list of Prof. Rienstra's publications can be found here.

"Solid-State NMR Study of the Charge-Transfer Complex between Ubiquinone-8 and Disulfide Bond Generating Membrane Protein DsbB", Tang, M.; Sperling, L.J.; Berthold, D.A.; Nesbitt, A.E.; Gennis, R.B.; Rienstra, C. M. J. Am. Chem. Soc.2011- in press.

"Straightforward, effective calibration of SPINAL-64 decoupling results in the enhancement of sensitivity and resolution of biomolecular solid-state NMR", Comellas, G.C.; Lopez, J.J.; Nieuwkoop, A.J.; Lemkau, L.R.; Rienstra, C.M. J. Magn. Reson.2011- in press[online].  

"GFT projection NMR spectroscopy for proteins in the solid state", Franks, W. T..; Atreya, H. S..; Szyperski, T.; Rienstra, C. M. J. Biomol. NMR201048(4), 213-223. [online] 

"Automated protein resonance assignments of magic angle spinning solid-state NMR spectra of b1 immunoglobulin binding domain of protein G (GB1)", Moseley, H. N. B.; Sperling, L. J.; Rienstra, C. M. J. Biomol. NMR201048, 123-128. [online] 

"Lipid-Protein Correlations in Nanoscale Phospholipid Bilayers Determined by Solid-State Nuclear Magnetic Resonance", Kijac, A.; Shih, A. Y.; Nieuwkoop, A. J.; Schulten, K.; Sligar, S. G.; Rienstra, C. M. Biochemistry2010,49, 9190-9198. [online] 

"High Resolution (13)C-Detected Solid-State NMR Spectroscopy of a Deuterated Protein", Tang, M.; Comellas, G.; Mueller, L. J.; Rienstra, C. M. J. Biomol. NMR201048, 103-111. [online] 

"Backbone 1H, 13C and 15N Resonance Assignments of the Extracellular Domain of Tissue Factor", Boettcher, J. M.; Clay, M. C.; Lahood, B. J.; Morrissey, J. H.; Rienstra, C. M. Biomol. NMR Assign.20104(2), 183-185. [online] 

"NMR Determination of Protein pKa Values in the Solid State", Frericks Schmidt, H. L.; Shah, G. J.; Sperling, L. J.; Rienstra, C. M. J. Phys. Chem. Lett.20101, 1623-1628. [online] 

"Supramolecular Protein Structure Determination by Site-specific Long-range Intermolecular Solid State NMR Spectroscopy", Nieuwkoop, A. J..; Rienstra C. M. J. Am. Chem. Soc.2010132(22), 7570-7571. [online]

"Assignment Strategies for Large Proteins by Magic-Angle Spinning NMR: The 21-kDa Disulfide-Bond-Forming Enzyme DsbA", Sperling, L. J.; Berthold, D. A.; Sasser, T. L.; Jeisy-Scott, V.; Rienstra, C. M. J. Mol. Biol.,2010399(2), 268-282. [online] 

"J-Based 3D Sidechain Correlation in Solid-State Proteins", Tian, Y.; Chen, L.; Niks, D.; Kaiser, J. M.; Lai, J.; Rienstra, C. M.; Dunn, M. F.; Mueller, L. J. Phys. Chem. Chem. Phys.200911(32), 7078-7086. [online] 

"Atomic Resolution Protein Structure Determination by Three-Dimensional Transferred Echo Double Resonance Solid-State Nuclear Magnetic Resonance Spectroscopy", Nieuwkoop, A. J.; Wylie, B. J.; Franks, W. T.; Shah, G. J.; Rienstra C. M. J. Chem. Phys.2009131(9), 095101. [online]

"Protein Structure Refinement Using 13Cα Chemical Shift Tensors", Wylie, B. J.; Schwieters, C. D.; Oldfield, E.; Rienstra C. M. J. Am. Chem. Soc.2009131(3), 985-992. [online]

"Proton-Detected Solid-state NMR of Natural Abundance Peptide and Protein Pharmaceuticals", Zhou, D. H.; Shah, G. J.; Mullen, C.; Sandoz, D.; Rienstra, C. M. Angew. Chem. 200948(7), 1253-1256. [online]

"Membrane-Induced Folding of the cAMP-Regulated Phosphoprotein Endosulfine-α", Boettcher, J. M.; Hartman, K. L.; Ladror, D. T.; Qi, Z.; Woods, W. S.; George, J. M.; Rienstra, C. M. Biochemistry 200847, 13257-12364. [online]

Awards

  • Founders Medal from the International Conferences on Magnetic Resonance in Biological Systems (ICMRBS), 2008
  • I. C. Gunsalus Scholar, UIUC, 2008-2009
  • Alfred P. Sloan Research Fellow, 2007-2009
  • Keck Foundation Research Excellence Award, 2006-2007
  • Excellence in Teaching Award, School of Chemical Sciences, 2005
  • National Center for Supercomputing Applications Fellow, 2005-2006
  • Cottrell Scholars Award, Research Corporation
  • UIUC Center for Advanced Study Fellow, 2005-2006
  • Analytical Chemistry Award, Eli Lilly
  • NSF CAREER Award
  • Research Innovation Award, Research Corporation
  • Petroleum Research Fund Award, ACS
  • National Institutes of Health Postdoctoral Fellowship
  • Howard Hughes Medical Institute Predoctoral Fellowship

Patents

Photo of Chad M. Rienstra