Biophysical Chemistry

Faculty currently associated with this research themes.

Biophysical Chemistry at the University of Michigan applies the quantitative methods of chemistry to biological problems. For instance, Biophysical Chemistry at UM includes:

Carlson Lab +

Computer simulations of protein-ligand molecular recognition

Biophysical chemistry in the Carlson lab involves computer simulations of protein-ligand complexes to understand protein flexibility, allostery, molecular recognition, and drug design. 

Link to Carlson Lab

Walter Lab +

Ribozyme and Spliceosome Mechanisms Dissected at the Single Molecule Level

Impact: RNA, in the form of both protein-coding messenger RNAs (mRNAs) and a plethora of recently discovered non-coding RNAs, plays a central role in human cell biology by catalyzing, guiding or coordinating all processes involved in the expression of the human genome. In fact, RNA profiling has revealed that ~75% of the human genome is transcribed into RNA in at least one cell type at some point. Understanding the structure-dynamics-function relationships of this vast universe of RNAs, in pursuit of both a fundamental understanding of life as we know it and new medicines against debilitating diseases such as cancer, requires new tools.

Objective: To study the structure-dynamics-function relationships of RNA enzymes (“ribozymes”) and the spliceosome, a mega-Dalton RNA-protein complex responsible for converting precursor-messenger RNAs into their mature mRNAs in all eukaryotes.

Approach: Using a combination of in vitro probing by single molecule fluorescence resonance energy transfer (FRET) and in silico single molecule molecular dynamics (MD) we are dissecting the mechanism of action and dynamics of both coding and non-coding RNAs, including various ribozymes and the yeast spliceosome.

Link to Walter Lab 

Lehnert Lab +

Second coordination sphere effects in heme proteins and flavodiiron nitric oxide reductases.

Cytochrome P450s (Cyt P450) are a super family of b-type heme-containing enzymes with a thiolate (cysteine) as a proximal ligand to the heme’s iron center. These enzymes generally activate dioxygen and catalyze a large variety of hydroxylation and oxidation reactions of diverse substrates. Closely related to the Cyt P450s are Nitric Oxide Synthases (NOS), which facilitate the bio¬synthesis of nitric oxide (NO) in mammals from L-arginine. The fascinating and perplexing versatility of all of these enzymes with respect to the reactions that they catalyze requires a subtle fine-tuning of the properties, electronic structures, and redox potentials of their {heme-thiolate} active sites. This is generally facilitated by second coordination sphere (SCS) interactions, in particular hydrogen bonding with the axial Cys ligand of heme. In this way, SCS effects allow principally similar {heme-thiolate} active sites to perform a surprisingly diverse range of functions. Despite this central importance of SCS effects, it has been found extremely challenging to establish rigorous structure-function relationships that allow for a quantitative evaluation of SCS effects. Our long term goal is to use detailed spectroscopic studies, in particular resonance Raman, electron paramagnetic resonance, magnetic circular dichroism, and nuclear resonance vibrational spectroscopy and DFT calculations to obtain a quantitative understanding of how SCS effects influence Fe-S bond strength, heme conformation, and heme propionate protonation state, and how this in turn deter¬mines functions in Cyt P450s and NOS.

Recently, similar studies were also initiated on the electron transfer protein Cytochrome c

Nitric oxide (NO) is a signaling molecule in mammals that plays a key role in blood pressure control and nerve signal transduction, and in addition, it is also produced by macrophages at micromolar concentrations as a key immune defense agent to kill invading pathogens. At such high concentrations, NO is able to inactivate important metalloenzymes, and this has been linked to NO toxicity against microbes. However, a number of pathogens (e.g., Helicobacter pylori, Neisseria meningitides, Salmonella enterica) have evolved defenses against NO toxicity by expressing flavodiiron NO reductases (FNORs). These enzymes are able to efficiently remove NO by reducing it to nontoxic N2O. FNORs therefore contribute to the infectious potential of pathogens, as they allow them to proliferate in the human body, and cause harmful chronic infections and inflammations. However, despite this significance for microbial pathogenesis, the mechanism of FNORs and how the flavodiiron active sites of these proteins mediate the degradation of NO is largely unknown. Our overall goal is to build a detailed mechanism for the binding, activation, and reduction of NO at the flavodiiron active site of FNORs using spectroscopic and theoretical methods. Such information will be crucial to developing agents for thwarting the action of FNORs.

Figure 1. MCD C-term spectrum of high-spin ferric Cytochrome P450cam Q360P measured at 5 K in phosphate buffer with 50 % (v/v) glycerol added. The colored lines represent a correlated Gaussian fit of these data, where the predominant polarizations are color-coded (xy-polarizations in blue, z-polarizations in green, and undetermined polarizations in gray). [M. G. I. Galinato, T. Spolitak, D. P. Ballou, N. Lehnert, Biochemistry 50 (2011) 1053-1069]

Link to Lehnert Lab

Ruotolo Lab +

Screening for Conformationally-Selective Protein/Inhibitor Interactions.

Impact: Proteins, and the macromolecular machines they collaborate to create, comprise the vast majority of targets of pharmaceutical interest.  Most technologies designed to search for new therapeutic small molecules are tuned only to find those that bind protein targets with the greatest affinity, or significantly alter enzyme function.  However, small molecules capable of modulating the function of dynamic protein assemblies, the causal factors in many human diseases, are unlikely to be among those that bind most tightly to proteins via classical enzyme active sites.  Similarly, many next-generation inhibitors must be selective for a specific protein conformation, as well as for a specific active site. Therefore, we are developing new technologies, capable of rapidly assessing protein conformation in the context of small molecule therapeutics, based on ion mobility-mass spectrometry (IM-MS).

Objective: We seek to apply our new conformatinally-selective IM-MS technology to cutting-edge screening efforts aimed at discovering new therapeutics for Alzheimer's disease and cancer.

Approach: Following MS selection according to m/z, activation is achieved by accelerating ions into an ion trap pressurized with argon.  Energetic collisions increase the internal temperature of the protein-ligand complex and illicit unfolding transitions which are measured by IM – which separates gas phase ions according to their shape and size ().  Since gas-phase protein unfolding is the primary component of these data, we term the output ‘collision induced unfolding’ (CIU).  At coincident energies, the protein also undergoes charge-stripping (through the dissociation of small, loosely-bound, positively-charged counter-ions) and ligand dissociation events that also contribute to the final CIU fingerprint.  We can use our fingerprint data to distinguish between kinase inhibitors having different conformational selectivities.   For example, dasatinib (Sprycel) is a non-selective kinase inhibitor with respect to protein conformation, while imatinib (Gleevec) interacts with only the inactive form (Figure B).  CIU Fingerprints can easily distinguish between these two binding modes, and can be extended in a high-throughput screening format to detect other conformationally-selective kinase binders.

Figure A and Figure B. 

Link to Ruotolo Lab

Kopelman Lab +

Asynchronous Cell Rotation: Magnetic Nanobeads for Cancer Cell Malignancy Determination

Biosensors with increasingly high sensitivity are crucial for probing small scale properties. The asynchronous magnetic bead rotation (AMBR) sensor is an emerging sensor platform, based on magnetically actuated rotation. Here the frequency dependence of the AMBR sensor’s sensitivity is investigated. An asynchronous rotation frequency of 145 Hz is achieved. This increased frequency will allow for a calculated detection limit of as little as a 59 nm change in bead diameter, which is a dramatic improvement over previous AMBR sensors and further enables physical and biomedical applications.

Link to Kopelman Lab

Ramamoorthy Lab +

Solid-state NMR to elucidate structure of membrane proteins, cancer metabolomics and bone.

Overview: High-resolution structure determination of biological systems that are difficult to study by traditional biophysical techniques is one of the most important and challenging aspects of modern scientific research. Solid-state NMR spectroscopy is an indispensable technique for the structural and dynamic characterization of immobile and non-crystalline proteins that are difficult to study by X-ray crystallography or by solution NMR.

Research Focus: Development of leading-edge NMR methods and their applications to elucidate the structure and understand the molecular dynamics of complex biological systems is the main focus of our research. A major emphasis is directed towards structural determination of the membrane-associated proteins and peptides such as cytochrome b5, myelin basic protein, antimicrobial peptides, toxin, and amyloid peptides. Another area of interest involves the application of NMR methods to investigate the metabolomics of cancer tissues. We also make intensive use of state-of-the-art solid-state magic-angle spinning (MAS) NMR techniques to investigate the atomic-scale structure of the protein-mineral interface in bone tissues in order to understand quantitatively the impact of water loss, aging, and diseases on the quality of bone and its susceptibility to fracture.

Link to Ramamoorthy Lab

Biteen Lab +

Single-Molecule Imaging of Protein Localization and Dynamics in Live Bacteria Cells.

Single-molecule fluorescence brings the resolution of optical microscopy down to the nanometer scale. This allows us to unlock the mysteries of how biomolecules work together in cells. We have developed novel methods for single-molecule investigations and apply them to three prokaryotic systems: membrane-bound transcription activation in Vibrio cholerae, carbohydrate catabolism in Bacteroides thetaiotaomicron, and DNA mismatch repair in Bacillus subtilis. Each presents unique challenges, and we discuss the adaptations developed for each system, including a comparison of membrane and soluble proteins, extensions to two-color and 3D imaging, and live anaerobic cell studies.

Link to Biteen Lab