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The Michigan Biophysics Graduate Program (MBGP) was established in 1949, making it one of the first such programs in the world. The intellectual base of the program was significantly broadened in the 1980 when faculty members from a number of other units on campus were invited to join. Currently over forty faculty members from a variety of disciplines participate as mentors for the Ph.D. students enrolled in the MBGP providing our students with rich opportunities for academic learning and research. The MBGP has two main objectives: 1) to provide graduate students with both the intellectual and technical training in modern biophysics, 2) to sensitize our students to the power and unique opportunities of interdisciplinary work and thinking so as to train them to conduct research that crosses the boundaries between the biological and physical sciences. The program offers students opportunities to conduct research in a variety of areas of contemporary biophysics including structural biology, single molecule spectroscopy, spectroscopy and its applications, computational biology, membrane biophysics, neurobiophysics and enzymology. The MBGP offers a balanced curriculum that aims to provide our students with a strong academic base and, at the same time, accommodate their different academic backgrounds. Judging its past performance through the success of its former students, the MBGP has been highly successful, and there is every reason to believe that strong training in the biophysical sciences, as provided by the MBGP, will become even more valuable in the future both in the academic and the industrial settings. in the academic and the industrial settings.

Five nonpeptide, small-molecule inhibitors of the human MDM2-p53 interaction are presented, and each inhibitor represents a new scaffold. The most potent compound exhibited a Ki of 110 +/- 30 nM. These compounds were identified using our multiple protein structure (MPS) method which incorporates protein flexibility into a receptor-based pharmacophore model that identifies appropriate hotspots of binding. Docking the inhibitors with an induced-fit docking protocol suggested that the inhibitors mimicked the three critical binding residues of p53 (Phe19, Trp23, and Leu26). Docking also predicted a new orientation of the scaffolds that more fully fills the binding cleft, enabling the inhibitors to take advantage of additional hydrogen-bonding possibilities not explored by other small molecule inhibitors. One inhibitor in particular was proposed to probe the hydrophobic core of the protein by taking advantage of the flexibility of the binding cleft floor. These results show that the MPS technique is a promising advance for structure-based drug discovery and that the method can truly explore broad chemical space efficiently in the quest to discover potent, small-molecule inhibitors of protein-protein interactions. Our MPS technique is one of very few ensemble-based techniques to be proven through experimental verification of the discovery of new inhibitors.

An increasing number of RNAs are being discovered that perform their functions by undergoing large changes in conformation in response to a variety of cellular signals, including recognition of proteins and small molecular targets, changes in temperature, and RNA synthesis itself. The measurement of NMR residual dipolar couplings (RDCs) in partially aligned systems is providing new insights into the structural plasticity of RNA through combined characterization of large-amplitude collective helix motions and local flexibility in noncanonical regions over a wide window of biologically relevant timescales (

Burkholderia cenocepacia is an important respiratory pathogen in persons with cystic fibrosis. We compared the proteomes of clinical and environmental isolates of B. cenocepacia by using a 2D liquid separation method coupled with mass spectrometry. Proteome maps of four B. cenocepacia isolates were generated. In the first dimension, 5 mg of protein from each isolate was fractionated by chromatofocusing (CF) in the range of pH 4.0-7.0. In the second dimension, each CF fraction was separated by NPS-RP-HPLC. Results of the 2D liquid separation were visualized as 2D UV maps, which allowed direct comparison of proteomes with high resolution and reproducibility. From the proteomic comparison of the four isolates, 38 of 96 differentially abundant proteins were identified by peptide mass fingerprinting and MS/MS sequence analysis using a partially annotated B. cenocepacia protein database. Many of the identified proteins in the clinical isolates are involved in gene translation and bacterial virulence such as transmissibility, resistance, and quorum sensing.

A comprehensive platform that integrates information from the protein and peptide levels by combining various MS techniques has been employed for the analysis of proteins in fully malignant human breast cancer cells. The cell lysates were subjected to chromatofocusing fractionation, followed by tryptic digestion of pH fractions for on-line monolithic RP-HPLC interfaced with linear ion trap MS analysis for rapid protein identification. This unique approach of direct analysis of pH fractions resulted in the identification of large numbers of proteins from several selected pH fractions, in which approximately 1.5 microg of each of the pH fraction digests was consumed for an analysis time of ca 50 min. In order to combine valuable information retained at the protein level with the protein identifications obtained from the peptide level information, the same pH fraction was analyzed using nonporous (NPS)-RP-HPLC/ESI-TOF MS to obtain intact protein MW measurements. In order to further validate the protein identification procedures from the fraction digest analysis, NPS-RP-HPLC separation was performed for off-line protein collection to closely examine each protein using MALDI-TOF MS and MALDI-quadrupole ion trap (QIT)-TOF MS, and excellent agreement of protein identifications was consistently observed. It was also observed that the comparison to intact MW and other MS information was particularly useful for analyzing proteins whose identifications were suggested by one sequenced peptide from fraction digest analysis., (Copyright 2007 John Wiley & Sons, Ltd.)

We describe the dielectric effects in a novel branched phthalocyanine system. The synthesis and characterization of the hyperbranched structure are provided. The dielectric constant was approximately 45 over many decades of frequency, and the dispersion was small up to 1 MHz. The losses experienced in this novel material were very small (close to 0.001) at high frequency. The mechanism of this novel effect in the branched structure involves a delocalized polaronic state which takes advantage of the strong intramolecular interactions in the system.

Using residual chemical shift anisotropies (RCSAs) measured in a weakly aligned stem-loop RNA, we examined the carbon chemical shift anisotropy (CSA) tensors of nucleobase adenine C2, pyrimidine C5 and C6, and purine C8. The differences between the measured RCSAs and the values back-calculated using three nucleobase carbon CSA sets [D. Stueber, D.M. Grant, 13C and 15N chemical shift tensors in adenosine, guanosine dihydrate, 2'-deoxythymidine, and cytidine, J. Am. Chem. Soc. 124 (2002) 10539-10551; D. Sitkoff, D.A. Case, Theories of chemical shift anisotropies in proteins and nucleic acids, Prog. NMR Spectrosc. 32 (1998) 165-190; R. Fiala, J. Czernek, V. Sklenar, Transverse relaxation optimized triple-resonance NMR experiments for nucleic acids, J. Biomol. NMR 16 (2000) 291-302] reported previously for mononucleotides (1.4 Hz) is significantly smaller than the predicted RCSA range (-10-10 Hz) but remains larger than the RCSA measurement uncertainty (0.8 Hz). Fitting of the traceless principal CSA values to the measured RCSAs using a grid search procedure yields a cytosine C5 CSA magnitude (CSAa=(3/2.(delta11(2)+delta22(2)+delta33(2)))1/2=173+/-21 ppm), which is significantly higher than the reported mononucleotide values (131-138 ppm) and a guanine C8 CSAa (148+/-13 ppm) that is in very good agreement with the mononucleotide value reported by solid-state NMR [134 ppm, D. Stueber, D.M. Grant, 13C and (15)N chemical shift tensors in adenosine, guanosine dihydrate, 2'-deoxythymidine, and cytidine, J. Am. Chem. Soc. 124 (2002) 10539-10551]. Owing to a unique sensitivity to directions normal to the base plane, the RCSAs can be translated into useful long-range orientational constraints for RNA structure determination even after allowing for substantial uncertainty in the nucleobase carbon CSA tensors.

The wild type form of Red fluorescent protein (DsRed), an intrinsically fluorescent protein found in tropical corals, is found to be highly selective, reversible and sensitive for both Cu(+) and Cu(2+), with a nanomolar detection limit. The selectivity towards these ions is retained even in the presence of other heavy metal ions. The K(d) values for monovalent and divalent copper, based on single binding isotherms, are 450 and 540 nM, respectively. The wild type DsRed sensitivity to Cu(2+) (below 1 ppb) is seven orders of magnitude better than that of the related wild type Green Fluorescent protein (GFP), and it is even 40 times more sensitive than engineered mutants of GFP. Potential binding sites have been proposed, based on amino acid sequences for copper binding and the distance from the chromophore, with the aid of computer modeling.

Topoisomerases are enzymes of quintessence to the upkeep of superhelical DNA, and are vital for replication, transcription and recombination. An atomic-resolution model for human topoisomerase I in covalent complex with DNA is simulated using molecular dynamics with external potentials that mimic torque and bias the DNA duplex downstream of a single-strand cut to rotate around the intact strand, according to the prevailing enzymatic mechanism. The simulations reveal the first dynamical picture of how topoisomerase accommodates large-scale motion of DNA as it changes its supercoiling state, and indicate that relaxation of positive and negative supercoils are fundamentally different. To relax positive supercoils, two separate domains (the 'lips') of the protein open up by about 10-14 A, whereas to relax negative supercoils, a continuous loop connecting the upper and lower parts (and which was a hinge for opening the lips) stretches about 12 A while the lips remain unseparated. Normal mode analysis is additionally used to characterize the functional flexibility of the protein. Remarkably, the same combination of low-frequency eigenvectors exhibit the dominant contribution for both rotation mechanisms through a see-saw motion. The simulated mechanisms suggest mutations to control the relaxation of either type of supercoiling selectively and advance a hypothesis for the debated role of the N-terminal domain in supercoil relaxation.

Four theoretical and computational approaches used at the University of Michigan to analyze NMR paramagnetic relaxation enhancement (NMR-PRE) are described. The primary objective of the theory is to describe the relationship of the NMR-PRE phenomenon to the electron spin hamiltonian and the spin energy level structure when zero field splitting interactions are significant. Four formulations of theory are discussed: (1) spin dynamics simulation; (2) the laboratory frame "constant H(S)" formulation; (3) the Molecular Frame "constant H(S)" formulation; and (4) the zfs-limit "constant H(S)" formulation. No single theoretical approach describes all important aspects of the relaxation mechanism in a fully satisfactory way. We use the four formulations in a complementary manner to provide as complete a picture of the relaxation mechanism as possible. We also discuss the integration of NMR-PRE theory and recently developed theory of electron spin relaxation which accounts for effects of the permanent zfs hamiltonian.

We present a computational study of barnase unfolding during import into mitochondria through a model translocon. In contrast to thermal (or chemical) unfolding, the major intermediates of co-translocational unfolding are mainly mediated by non-native interactions accompanying the protein configurations induced by pulling forces. These energy contributions, combined with backbone topological constraints imposed by the model pore, result in milestones along the unfolding pathways which are significantly different not only from those experienced during thermal (or chemical) denaturation, but also from those observed in single-molecule pulling by both ends without pore constraints. Two on-pathway major translocation intermediates trapped in long-lived states by significantly high unfolding barriers are identified. A fraction of these pathways can, however, skip such local kinetic traps and result in extremely fast translocations, leading to a dramatic kinetic partitioning spanning approximately four orders of magnitude. The fraction of fast translocation events is shown to increase upon switching the pull off and on, when compared to pulling at constant force. This suggests a "catalytic" mechanism by which the mitochondrial import machinery regulates this partitioning by repetitively pulling in cycles. A number of mutation sites that alter the kinetic "flow" of the unfolding trajectories are suggested and tested.

During development of the neuromuscular junction (NMJ), extrajunctional expression of genes, whose products are destined for the synapse, is suppressed by muscle activity. One of the best-studied examples of activity-dependent gene regulation in muscle are those encoding nicotinic acetylcholine receptor (nAChR) subunits. We recently showed that nAChR gene expression is inhibited by calcium/calmodulin-dependent protein kinase II (CaMKII) and CaMKII inhibitors block activity-dependent suppression of these genes. Here we report results investigating the mechanism by which CaMKII suppresses nAChR gene expression. We show that the muscle helix-loop-helix transcription factor, myogenin, is necessary for activity-dependent control of nAChR gene expression in cultured rat myotubes and is a substrate for CaMKII both in vitro and in vivo. CaMKII phosphorylation of myogenin is induced by muscle activity and this phosphorylation influences DNA binding and transactivation. Thus we have identified a signaling mechanism by which muscle activity controls nAChR gene expression in developing muscle.

Regulators of G-protein signaling (RGS) proteins form a multifunctional signaling family. A key role of RGS proteins is binding to the G-protein Galpha-subunit and acting as GTPase-activating proteins (GAPs), thereby rapidly terminating G protein-coupled receptor (GPCR) signaling. Using the published RGS4-Gialpha1 X-ray structure we have designed and synthesized a series of cyclic peptides, modeled on the Gialpha Switch I region, that inhibit RGS4 GAP activity. These compounds should prove useful for elucidating RGS-mediated activity and serve as a starting point for the development of a novel class of therapeutic agent.

A molecular weight map of the protein content of ES2 human clear cell ovarian carcinoma cells has been produced using a two-dimensional (2-D) liquid separations/mass mapping technique. This method uses a 2-D liquid separation of proteins from whole cell lysates coupled on-line to an electrospray ionization-time of flight (ESI-TOF) mass spectrometer to map the accurate intact molecular weight (M(r)) of the protein content of the cells. The two separation dimensions involve the use of liquid isoelectric focusing as the first phase and nonporous silica reversed-phase high-performance liquid chromatography (HPLC) as the second phase of separation. The detection by ESI-TOF-MS provides an image of pI versus M(r) analogous to 2-D gel electrophoresis. Each protein is then identified based upon matrix-assisted laser desorption/ionization (MALDI)-TOF-MS peptide mapping and intact M(r) so that a standard map is produced against which other ovarian carcinoma cell lines can be compared. The accurate intact M(r) together with the pI fraction, and peptide map serve to tag the protein for future interlysate comparisons. An internal standard is also used to provide a means for quantitation for future interlysate studies. In the ES2 cell line under study it is shown that nearly 900 M(r) bands are detected over 17 pI fractions from pH 4 to 12 and a M(r) range up to 85 kDa and that around 290 of these bands can be identified using mass spectrometric based techniques. The protein M(r) is detected within an accuracy of 150 ppm and it is shown that many of the proteins in this human cancer sample are modified compared to the database. The protein M(r) map may serve as a highly reproducible standard Web-based method for comparing proteins from related human cell lines.

Electrophilic fluorination of enantiomerically pure 2-pyrrolidinones (4) derived from (L)-glutamic acid has been investigated as a method for the synthesis of single stereoisomers of 4-fluorinated glutamic acids. Reaction of the lactam enolate derived from 9 with NFSi results in a completely diastereoselective monofluorination reaction to yield the monocyclic trans-substituted alpha-fluoro lactam product 21. Unfortunately, a decreased kinetic acidity in 21 and other structurally related monofluorinated products renders them resistant to a second fluorination. In contrast, the bicyclic lactam 12 is readily difluorinated under the standard conditions described to yield the alpha,alpha-difluoro lactam 24. The difference in reactivity between the two types of related lactams is attributed mainly to the presence or lack of a steric interaction between the base used for deprotonation and the protecting group present in the pyrrolidinone substrates. This conclusion was reached based on analysis of the X-ray crystal structure of 21, molecular modeling, and experimental evidence. The key intermediates 21 and 24 are converted to (2S,4R)-4-fluoroglutamic acid and (2S)-4,4-difluoroglutamic acid, respectively.

The Old Yellow Enzyme has been shown to catalyze efficiently the NADPH-linked reduction of nitro-olefins. The reduction of the nitro-olefin proceeds in a stepwise fashion, with formation of a nitronate intermediate that is freely dissociable from the enzyme. The first step involves hydride transfer from the enzyme-reduced flavin to carbon 2 of the nitro-olefin. The protonation of the nitronate at carbon 1 to form the final nitroalkane product also is catalyzed by the enzyme and involves Tyr-196 as an active site acid/base. This residue also is involved in aci-nitro tautomerization of nitroalkanes, the first example of a nonredox reaction catalyzed by the enzyme.

Factor VIII and von Willebrand factor (vWF) circulate in the plasma as a noncovalent protein complex. Circulating levels of factor VIII are coordinately regulated with circulating levels of vWF in which the ratio is maintained at 1 molecule of factor VIII for 50 to 100 vWF subunits. Infusion of vWF into vWF-deficient animal models and human patients yields a secondary increase in circulating levels of factor VIII. We have studied the mechanism of the secondary rise in factor VIII in a porcine model of vWF deficiency. On infusion of vWF into a vWF-deficient pig there was an approximately fivefold increase in circulating factor VIII activity. Liver biopsies were taken pre- and post-vWF infusion for isolation of total messenger RNA (mRNA). Factor VIII-specific mRNA was measured by an RNAse protection assay. The results showed no difference in the liver-specific factor VIII mRNA on vWF infusion. These results indicate that the secondary rise in factor VIII levels in response to exogenous vWF infusion is not dependent on increased steady-state levels of factor VIII mRNA in the liver.

Given the importance of tyrosine phosphorylation of proteins in signalling pathways, it is perhaps not surprising that protein tyrosine phosphatases (PTPs) are involved in the pathogenesis of certain human diseases. A PTP produced by the Yersinia bacteria (which can cause bubonic plague, septicemia and enteric diseases) is thought to be used as a 'weapon' against host cell functions. In addition, dysfunction of cells' endogenous PTPs may contribute to defective immune function, to cancer and to diabetes.

The NMR paramagnetic relaxation enhancement (NMR-PRE) produced in the solvent proton resonance by manganese in the S0 and S2 states of the oxygen evolving center (OEC) has been recorded for three Photosystem II (PS II)-enriched preparations: (1) PS II-enriched thylakoid membrane fragments (TMF-2 particles); (2) salt-washed (2M NaCl) TMF-2 particles; and (3) the octylglucopyranoside (OGP)-solubilized PS II complex. The second and third preparations, but not the first, are depleted of the peripheral 17 and 23 kD polypeptides associated with the OEC. It has been proposed that depletion of these polypeptides increases the exposure of OEC manganese to the aqueous phase. The NMR-PRE response measures the quantity (T1m+τm)(-1), where T1m is the spin relaxation time and τm is the mean residence time with respect to chemical exchange reactions of solvent protons in the manganese coordination sphere, and, thus, the NMR-PRE provides a direct measure of the solvent proton chemical exchange rate constant τm (-1). This study tested whether the 17 and 23 kD polypeptides shield the OEC from the solvent phase and whether their depletion enhances the S2 and S0 NMR-PRE signals by removing a kinetic barrier to the solvent proton chemical exchange reaction. The amplitude of the S2 NMR-PRE signal, measured in its chemical exchange-limited regime (τm>T1m), is slightly decreased, rather than increased, in preparations (2) and (3) relative to (1), indicating that removal of the 17 and 23 kD polypeptides slightly slows, rather than accelerates, the rate-limiting steps of the solvent proton chemical exchange reactions. In addition, the lifetime of the S2 state was shortened several-fold in the solubilized PS II complex and in salt-washed TMF-2 membranes relative to untreated TMF-2 control samples. The S0 NMR-PRE signal, which is present in TMF-2 suspensions, was not detected in suspensions of the solubilized PS II complex, even though these samples contained high concentrations of active manganese centers (approximately double those of the TMF-2 control) and exhibited an S2 NMR-PRE signal of comparable amplitude to that of the TMF-2 preparation. These results suggest that the 17 and 23 kD extrinsic polypeptides do not shield the NMR-visible water binding site in the OEC from the aqueous phase, although their removal substantially alters the proton relaxation efficiency by shortening T1m.