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.

 

 

 

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.

 

Tunable Fluorescent Reporters

In vivo optical imaging must contend with the limitations imposed by the optical window of tissue, 600 to 1000 nm. Although a wide array of fluorophores are available that are visualized in the red and near-IR region of the spectrum, with the exception of proteases, there are few long wavelength probes for enzymes. This situation poses a particular challenge for studying the intracellular biochemistry of erythrocytes, the high hemoglobin content of which optically obscures subcellular monitoring at wavelengths less than 600 nm.

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To address this, researchers in the Lawrence Group, published in Angewandte Chemie, developed tunable fluorescent reporters for protein kinase activity. The probing wavelength is preprogrammed by using readily available fluorophores, thereby enabling detection within the optical window of tissue, specifically in the far-red and near-IR region. These agents were used to monitor endogenous cAMP-dependent protein kinase activity in erythrocyte lysates and in intact erythrocytes when using a light-activatable reporter.

 

Representative Publications

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.

Low Copy Numbers of DC-SIGN in Cell Membrane Microdomains: Implications for Structure and Function. Ping Liu, Xiang Wang, Michelle S. Itano, Aaron K. Neumann, Aravinda M. de Silva, Ken Jacobson, Nancy L. Thompson. Traffic, Volume 15, Issue 2, pages 179–196, February 2014.

The Cellular Environment Stabilizes Adenine Riboswitch RNA Structure. Jillian Tyrrell, Jennifer L. McGinnis, Kevin M. Weeks, and Gary J. Pielak . Biochemistry, Article ASAP, DOI: 10.1021/bi401207q.

Impact of Reconstituted Cytosol on Protein Stability. Mohona Sarkar, Austin E. Smith, and Gary J. Pielak. Published online before print, November 11, 2013, doi: 10.1073/pnas.1312678110 PNAS November 11, 2013.

Molecular Basis for pH-Dependent Mucosal Dehydration in Cystic Fibrosis Airways. Alaina L. Garlanda, William G. Waltonb, Raymond D. Coakley, Chong D. Tan, Rodney C. Gilmore, Carey A. Hobbs, Ashutosh Tripathy, Lucy A. Clunes, Sompop Bencharit, M. Jackson Stutts, Laurie Betts, Matthew R. Redinbo, and Robert Tarran. PNAS, September 16, 2013, doi: 10.1073/pnas.1311999110.

β-Turn Sequences Promote Stability of Peptide Substrates for Kinases Within the Cytosolic Environment. Shan Yang, Angela Proctor, Lauren L. Cline, Kaiulani M. Houston, Marcey L. Waters and Nancy L. Allbritton. Analyst, 2013,138, 4305-4311.