Department of Chemistry

Biochemistry & Chemical Biology

Research ImageGraduate students in biochemistry and chemical biology meld molecular and structural biology with physical, organic and analytical chemistry to understand the molecular basis of biological processes and of human disease. Research in the Biochemistry and Chemical Biology Division focuses on the structure and function of proteins, membranes, DNA, RNA, large macromolecular complexes and viruses, natural product biogenesis, synthetic biology, and genomics.

Students are a constant source of new hypotheses for mechanisms underlying cellular machines like the ribosome and spliceosome, and for the protein and RNA folding problems. Students tackle these problems using biochemical methods, chemical biosensor technologies, protein and nucleic acid crystallography, in vitro and in vivo evolution, multi-dimensional NMR spectroscopy, surface chemistry, atomic force microscopy, fluorescence spectroscopy, and high-resolution mass spectrometry.

Doctoral students in Biochemistry and Chemical Biology leave the Department broadly trained for leadership roles in academia and industry.

 

 

 

Protein Crowder Charge and Stability

Macromolecular crowding effects arise from steric repulsions and weak, nonspecific, chemical interactions. Steric repulsions stabilize globular proteins, but the effect of chemical interactions depends on their nature. Repulsive interactions such as those between similarly charged species should reinforce the effect of steric repulsions, increasing the equilibrium thermodynamic stability of a test protein. Attractive chemical interactions, on the other hand, counteract the effect of hard-core repulsions, decreasing stability.

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Mohona Sarkar and Joe Lu, researchers in the Pielak Group, tested these ideas, published in Biochemistry, by using the anionic proteins from Escherichia coli as crowding agents and assessing the stability of the anionic test protein chymotrypsin inhibitor 2 at pH 7.0. The anionic protein crowders destabilize the test protein despite the similarity of their net charges. Thus, weak, nonspecific, attractive interactions between proteins can overcome the charge–charge repulsion and counterbalance the stabilizing effect of steric repulsion.

 

Strategies for Protein NMR

In-cell NMR spectroscopy, one of the pioneers of which is the Pielak Group here at Carolina Chemistry, provides insight into protein conformation, dynamics, and function at atomic resolution in living cells. Systematic evaluation of isotopic-labeling strategies is necessary to observe the target protein in the sea of other molecules in the cell.

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In a collaboration with scientists from the Chinese Academy of Sciences, published in Biochemistry, researchers in the Pielak Group investigate the detectability, sensitivity, and resolution of in-cell NMR spectra of the globular proteins GB1, ubiquitin, calmodulin, and bcl-xl-cutloop, resulting from uniform 15N enrichment, with and without deuteration, selective 15N-Leu enrichment, 13C-methyl enrichment of isoleucine, leucine, valine, and alanine, fractional 13C enrichment, and 19F labeling. Most of the target proteins can be observed by 19F labeling and 13C enrichment with direct detection because selectively labeling suppresses background signals and because deuteration improves in-cell spectra. The group's results demonstrate that the detectability of proteins is determined by weak interactions with intercellular components and that choosing appropriate labeling strategies is critical for the success of in-cell protein NMR studies.

 

Representative Publications

Local Iontophoretic Administration of Cytotoxic Therapies to Solid Tumors. James D. Byrne, Mohammad N. R. Jajja, Adrian T. O’Neill, Lissett R. Bickford, Amanda W. Keeler, Nabeel Hyder, Kyle Wagner, Allison Deal, Ryan E. Little, Richard A. Moffitt, Colleen Stack, Meredith Nelson, Christopher R. Brooks, William Lee, J. Chris Luft, Mary E. Napier, David Darr, Carey K. Anders, Richard Stack, Joel E. Tepper, Andrew Z. Wang, William C. Zamboni, Jen Jen Yeh, and Joseph M. DeSimone. Sci Transl Med 4 February 2015: Vol. 7, Issue 273, p. 273ra14.

Quinary Structure Modulates Protein Stability in Cells. William B. Monteith, Rachel D. Cohen, Austin E. Smith, Emilio Guzman-Cisneros, and Gary J. Pielak. PNAS, Early Edition, doi 10.1073 pnas.1417415112 .

Cell-Mediated Assembly of Phototherapeutics. Weston J. Smith, Nathan P. Oien, Robert M. Hughes, Christina M. Marvin, Zachary L. Rodgers, Junghyun Lee and David S. Lawrence. Angewandte Chemie International Edition, Volume 53, Issue 41, pages 10945-10948, October 6, 2014.

Optogenetic Engineering: Light-Directed Cell Motility. Robert M. Hughes and David S. Lawrence. Angewandte Chemie International Edition, Volume 53, Issue 41, pages 10904-10907, October 6, 2014.

RNA Motif Discovery by SHAPE and Mutational Profiling (SHAPE-MaP). Nathan A Siegfried, Steven Busan, Greggory M Rice, Julie A E Nelson & Kevin M Weeks. Nature Methods 11, 959–965 (2014).

Residue Level Quantification of Protein Stability in Living Cells. William B. Monteith and Gary J. Pielak. PNAS July 21, 2014, doi: 10.1073/pnas.1406845111 .

Nitric Oxide-Releasing Quaternary Ammonium-Modified Poly(amidoamine) Dendrimers as Dual Action Antibacterial Agents. Brittany V. Worley , Danielle L. Slomberg , and Mark H. Schoenfisch. Bioconjugate Chem., 2014, 25 (5), pp 918–927.

Protein Crowder Charge and Protein Stability. Mohona Sarkar, Joe Lu, and Gary Pielak. Biochemistry, 2014, 53 (10), pp 1601–1606.

Strategies for Protein NMR in Escherichia coli. Guohua Xu, Yansheng Ye, Xiaoli Liu, Shufen Cao, Qiong Wu, Kai Cheng, Maili Liu, Gary J. Pielak, and Conggang Li. Biochemistry, 2014, 53 (12), pp 1971–1981.

Long-Wavelength Fluorescent Reporters for Monitoring Protein Kinase Activity. Nathan P. Oien, Luong T. Nguyen, Dr. Finith E. Jernigan, Prof. Melanie A. Priestman and Prof. David S. Lawrence. Article first published online: 6 MAR 2014, DOI: 10.1002/anie.201309691.