| James Kempf
Assistant Professor, Department of Chemistry and Chemical Biology
Rensselaer Polytechnic Institute
Education:
Ph.D., California Institute of Technology, 2000
B.S., SUNY Fredonia, 1993
Career Highlights:
Prof. Kempf trained in chemical physics with Prof. Dan Weitekamp at Caltech, where he developed optical nuclear magnetic resonance (NMR) methods that he used to image single-electron wavefunctions in structured semiconductor materials. In 2001, he was honored with Caltech’s McCoy award for top doctoral research in chemistry. Following his graduate work, Dr. Kempf worked for one-year at Cornell with Prof. John Marohn. There he developed a nanoscale imaging technique using force-detected NMR, an approach that dramatically improves on the sensitivity of the conventional, inductively detected experiment. Just prior to his arrival at RPI, Dr. Kempf was an NIH Kirchstein postdoctoral fellow at Yale working with Prof. Pat Loria. There, he characterized relations between motional dynamics and the function of large (>50 kDa) protein molecules. At Yale, Dr. Kempf developed an NMR relaxation experiment that extends the timescale of accessible motions by an order of magnitude. In separate work, he uncovered surprising functional significance of motions in the enzyme triosephosphate isomerase even though the motions are 104‑fold faster than catalysis. This demonstrates the utility of this motional time scale as a reporter on the functional state of an enzyme.
Research Areas:
Our group pursues two main branches of research that share two common threads, NMR spectroscopy and functional characterization of biomolecular systems. The two areas differ in the biochemical applications and in our spectroscopic goals and approach. One, NMR Stark Spectroscopy, entails development of a novel solid-state NMR technique to characterize the electrostatic environment within proteins and its dramatic influence function. The second area uses cutting-edge, but well-developed, solution NMR methods to correlate intramolecular dynamics with protein function. Details on these branches are given below, along with particular sub-interests, motivations and goals.
Intramolecular Electrostatics from NMR Stark Spectroscopy
The distribution of charges (electrostatics) within biomolecular systems are dominant forces in function. Yet few experimental techniques are available to measure intramolecular properties indicative of local electrostatics, while computational estimates, though valuable, are often granted unwarranted confidence for lack of alternative techniques.
New, broadly applicable protocols are needed to measure electrostatic features in molecular contexts. Ideally, such methods will directly measure intramolecular charge distributions for correlation with functional behavior, while also providing checks on computational accuracy and guidance for theoretical advances. NMR, with its atomic-scale capabilities for nondestructive, noninvasive spectroscopy and imaging, seems well suited to such goals. However, the well-known influence of intramolecular electrostatics on NMR spectra is poorly understood, while direct measure of the effects of applied E fields is impractical.
We are developing a solid-state NMR method that will directly measure E-field induced changes in NMR spectra (NMR Stark effects) as a non-invasive, detailed probe of the intramolecular electrostatic environment. This promises more general access than available from current methods, which rely on local, non-native vibrational probes in proteins. In contrast, NMR Stark spectroscopy can provide greater detail using widely distributed native chemical groups (carbonyls and amides). The new technique may also serve as a structural tool by characterizing NMR Stark responses as a function of conformation. This will further advance solid-state NMR for structural characterization, an essential goal in study of biomolecular systems, such as membrane proteins, that are poorly amenable to other techniques.
NMR Study of Biomolecular Dynamics in Chemical Function
Functional behavior of biological macromolecules is often explicable only by considering motional dynamics. This requires that we step beyond traditional perspectives of static structures. Elucidating the roles of motion and structural flexibility will move us towards the long-term goals of enhanced therapeutic and commercial exploitation of biochemical processes ranging from enzyme catalysis to signal transduction. The grander goal of mechanistic understanding also requires a powerful, and uncommon, combination of dynamic and structural techniques. In this regard, NMR is the complete package. It delivers near-continuous time resolution across 15 orders of magnitude (ps to hours) and casts that view in a whole-molecule, atomic-resolution structural context.
Our group combines spectroscopic expertise with biochemical insight to understand complex dynamic phenomena in biological macromolecules. Biochemistry and molecular biology are also central to this research, while the statistical mechanics, thermodynamics and quantum-mechanical NMR evolution are essential for physical interpretation of experimental observations.
Current projects in our group explore the effects of post-translational modifications on protein function, in particular, by testing our hypothesis that these modifications modulate function via motional dynamics. Such intramolecular communication is referred to as allostery. We place particular emphasis on glycosylation, the attachment of an oligosaccharide to a protein. Glycosylation is the most frequent and varied of post-translational modifications, and yet it is the least-understood and most-poorly characterized because of the difficulty of glycoprotein production and the complexity of the oligosaccharide. Our group is working to develop new preparatory techniques, as well as dynamic and structural NMR methods to detail functional variation in these heterogeneous molecules.
- Glycosylation & Allosteric Control in RNase B
RNase B is a reduced-activity, glycosylated variant of the prototypical enzyme, RNase A. The oligosaccharide is anchored at the non-catalytic residue, Asn34, which is distant from the active site. Thus, the reason for reduced RNase B activity is uncertain. However, motional dynamics play a significant role in RNase A activity and evidence suggests that RNase B is rigidified. This motivates our hypothesis that glycosylation allosterically modulates RNase activity by altering polypeptide dynamics. We use NMR relaxation experiments to probe differential RNase A and B dynamics and their relation to function. Recent results reveal that glycosylation yields correlated modulation of both the catalytic rate and intramolecular motions with a known functional role.
- Modulation of Human Adult Hemoglobin (Hb A) by Glycation
Hb A provides the classic example of the cooperative behavior often observed among the active sites of multimeric proteins. In spite of extensive study, several aspects of Hb A activity remain mysterious, but a fresh opportunity for their elucidation is now available. Two mechanisms of O2 binding cooperativity in Hb A, with either sequential or concerted O2 binding, identically predict observed behavior. Quantifying solution-state, native motions by dynamic NMR can reveal the true mechanism to yield valuable insight to Hb A’s vital function. In addition, we are interested in modulation of Hb A motion and activity by the ambient chemical addition of glucose. This process, known as glycation, is significant in all adult humans and especially prevalent among diabetics, leading to secondary disease complications. By characterizing the dynamics of unmodified and glycated Hb A, we aim to understand the deleterious effects of glycation on O2 transport.
Selected Publications:
J.G. Kempf, M.A. Miller & D.P. Weitekamp, “Imaging Quantum Confinement with Optical and POWER NMR,” PNAS (in press, 2008).
J.G. Kempf, et al, “An Optical NMR Spectrometer for Larmor-beat Detection and High-Resolution POWER NMR,” Rev. Sci. Instrum. 79(6), 063904-12 (2008).
J.G. Kempf, J. Jung, C.M. Ragain, N.S. Sampson & J.P. Loria, “Dynamic Requirements for a Functional Protein Hinge,” J. Mol. Biol. 368(1), 131-49 (2007).
E. Kovrigin, J.G. Kempf, M. Grey & J.P. Loria, “Faithful Estimation of Dynamics Parameters from CPMG Relaxation Dispersion Measurements,” J. Magn. Reson. 18(1), 93-104 (2006).
Kempf, J.G., Loria, J.P., "Measurement of Intermediate Exchange Phenomena," Chapter 12 (pp181-226) in Protein NMR Techniques, vol. 278, Meth. Molec. Biol., Ed. by K. Downing (Humana Press, Totowa, NJ, 2004).
Kempf J.G., Jung J., Sampson, N.S., Loria, J.P., "Off-resonance TROSY (R1 – R1r) for quantitation of fast exchange in large proteins," J. Amer. Chem. Soc. 125 (40), 12064-5 (2003).
Kempf J.G., Loria J.P., "Protein dynamics from solution NMR – Theory and applications," Cell Biochemistry & Biophysics 37 (3), 187-211 (2003).
Kempf, J.G., Marohn, J.A. “Nanoscale Fourier Transform Imaging with Magnetic Resonance Force Microscopy,” Phys. Rev. Lett. 90 (8), 087601-4 (2003).
Kempf, J.G., Weitekamp, D.P. “Method for atomic-layer-resolved measurement of polarization fields by nuclear magnetic resonance,” J. Vac. Sci. Technol. B 18 (4), 2255-62 (2000).
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