Thesis title: "Influence of internal genome pressure for viral particle infectivity and stability"
Defense date: April 2015
Genome packaging during virus replication is an ATP-dependent process, resulting in a thermodynamically unstable state of packaged viral DNA. This energetically unfavorable confinement of the microns-long DNA molecule creates a large internal pressure inside the virus. The effects of tight genome confinement has previously been studied for multiple bacterial viruses. Recently, using a novel experimental assay, we have provided the first measurement of this DNA pressure within a eukaryotic herpesvirus, HSV-1. Dave's project focused on calorimetric investigations of internal pressure for specific viral systems, as well as comparative studies between evolutionarily diverse viruses. This experimental approach can uncover general physical properties of viruses that regulate viral infectivity.
B.S. Biology; B.S. Physics, Rowan University, Glassboro, NJ
Research Scientist at Cybergenetics in Pittsburgh, PA
PhD Advisor: Dr Alex Evilevitch
- D. W. Bauer, A. Evilevitch. Influence of internal DNA pressure on stability and infectivity of phage lambda. Journal of Molecular Biology. 427 (20), 3189–3200 (2015)
- D. W. Bauer, D. Li, J. Huffman, Fred Homa, K. Wilson, J. Leavitt, S. Casjens, J. Baines, A. Evilevitch. Exploring the balance between DNA pressure and capsid stability in Herpesviruses and phages. Journal of Virology. 89 (18), 9288–9298 (2015)
- D. W. Bauer, J. B. Huffman, F. L. Homa, A. Evilevitch. (2013) Herpes Virus Genome, The Pressure Is On. J. Am. Chem. Soc. 135, 11216-21
Thesis Title: Solution NMR studies of HIV-1 reverse transcriptase
Graduation Date: Nov 21, 2016
Naima used solution NMR as a method to study protein structure and dynamics. Standard NMR experiments use protein uniformly labeled with NMR active nuclei, such as 15N, 13C and 2H. Each nucleus reports on its surrounding environment and the ability to obtain structural information depends on being able to resolve, and unambiguously assign all resonance frequencies to unique nuclei. However, for large proteins (> 30 kDa) resonance overlap properties render this process difficult. Naima's goal was to help develop NMR methods to study large proteins in solution. Towards this goal, she applied selective labeling methods to study the structure and dynamics of the 118 kDa HIV-1 reverse transcriptase, a major drug target in the treatment of HIV-1 infection. The NMR spectra of RT produced by these labeling methods contain less resonance overlap since only a few residues are labeled.
Chemistry (biochemistry track) University of North Carolina at Chapel Hill
PhD Advisor: Dr Angela Gronenborn
Current Location: Postdoctoral associate in the Rees lab at CalTech / HHMI.
- Sharaf, N. G.; Barnes, C. O.; Charlton, L. M.; Young, G. B.; Pielak, G. J., A bioreactor for in-cell protein NMR. J. Magn. Reson., 202 (2010) 140-146.
- Miklos AC, Li C, Sharaf NG, Pielak GJ. 2010. Volume exclusion and soft interaction effects on protein stability under crowded conditions. Biochemistry 49: 6984-6991.
- Sharaf NG, Poliner E, Slack RL, Christen MT, Byeon IJ, Parniak MA, Gronenborn AM, Ishima R. The p66 immature precursor of HIV-1 reverse transcriptase. Proteins. 2014; 82(10):2343-52
- Sharaf, N. G., and Gronenborn, A. M. 19F-Modified Proteins and 19F-Containing Ligands as Tools in Solution NMR Studies of Protein Interactions. Methods in Enzymology 2015; 565:67-95
- Sharaf NG, Ishima R, Gronenborn AM. Conformational Plasticity of the NNRTI-Binding Pocket in HIV-1 Reverse Transcriptase: A Fluorine Nuclear Magnetic Resonance Study. Biochemistry. 2016 Jul 19;55(28):3864-73>
- Sharaf NG, Brereton AE, Byeon IL, Andrew Karplus P, Gronenborn AM. NMR structure of the HIV-1 reverse transcriptase thumb subdomain. J Biomol NMR. 2016 66(4):273-280
- Sharaf NG, Xi Z, Ishima R, Gronenborn AM. The HIV-1 p66 homodimeric RT exhibits different conformations in the binding-competent and -incompetent NNRTI site. Proteins. 2017 doi: 10.1002/prot.25383 (in press)
Inventor in a provisional patent titled: Device for particulate NMR samples in a fluid.
Thesis Title: Investigations of the structural changes in proapoptotic peroxidase- active cardiolipin-bound cytochrome c and liquid-gel phase transitions in liposomes using solid state NMR spectroscopy
Graduation Date: November 30, 2016
Abhishek performed structural and dynamical measurements on cytochrome c (cyt-c) in its lipid-bound state. Cyt-c is primarily known for its role in the electron transfer pathway but it also plays an important role in intrinsic apoptosis. A required step in this process is the peroxidation of mitochondrial lipid cardiolipin (CL). A crucial open question is regarding the structural changes in the bound protein that allow its covalently bound heme to catalyze the peroxidation of CL acyl chains in the membrane core. He used solid-state NMR methodology to study the structure and dynamics of both the CL bound cyt-c as well as the vesicles themselves using samples mimicking the proapoptotic conditions that trigger cyt-c’s peroxidase activity. These methods were complemented with various spectroscopic methods, including fluorescence based peroxidase assays, to correlate the function and structure of the bound cyt-c.
Bachelor’s of Science in Biochemistry, Temple University, Philadelphia, 2011
PhD Advisor: Dr. Patrick CA van der Wel
Current Location: Analyst with Highmark Inc., Pittsburgh, PA
- Mandal, A., Hoop, C.L., DeLucia, M., Kodali, R., Kagan, V.E., Ahn, J., & van der Wel, P.C.A. (2015) Structural changes and pro-apoptotic peroxidase activity of cardiolipin-bound mitochondrial cytochrome c. Biophys J. 109(9):1873-84
- Hoop CL, Lin HK, Kar K, Magyarfalvi G, Lamley JM, Boatz JC, Mandal A, Lewandowski JR, Wetzel R, van der Wel PC. Huntingtin exon 1 fibrils feature an interdigitated β-hairpin-based polyglutamine core. Proc Natl Acad Sci U S A. 2016;113(6):1546-51
- Merg AD, Boatz JC, Mandal A, Zhao G, Mokashi-Punekar S, Liu C, Wang X, Zhang P, van der Wel PC, Rosi NL. Peptide-Directed Assembly of Single-Helical Gold Nanoparticle Superstructures Exhibiting Intense Chiroptical Activity. J Am Chem Soc. 2016 in press
- Mandal A, van der Wel PC. MAS 1H NMR Probes Freezing Point Depression of Water and Liquid-Gel Phase Transitions in Liposomes. Biophys J. 2016; 111(9):1965-1973
- Mandal A, Boatz JC, Wheeler T, Van der Wel PC On the use of ultracentrifugal devices for routine sample preparation in biomolecular magic-angle-spinning NMR J. Biomol. NMR 2017 in press
Thesis title: Molecular Structural Insights of Polyglutamine-rich Amyloid-like Fibrils using UV Resonance Raman Spectroscopy
Graduation date: June 2016
MBSB PhD Advisor: Dr Sanford Asher (Dept. Chemistry; University of Pittsburgh)
David worked on the development of UV Resonance Raman (UVRR) spectroscopic methods to study amyloid fibril structure and aggregation kinetics. UVRR is exquisitely sensitive to protein/peptide conformations and can be used to selectively probe the electronic structure of UV-absorbing chromophores.
His my main research focus was the aggregation mechanism(s) of polyglutamine (polyQ) peptides and how they lead to amyloid fibril formation. There are at least nine neurodegenerative diseases associated with expansions in polyQ repeat segments of proteins. These simple polyQ peptides are useful model systems for studying the fundamental biophysics of larger, more complicated polyQ rich protein systems. Another focus was on the development of instrumentation for use in studying new biological systems with UVRR. For this, he worked on creating a high-resolution, high-throughput UV double monochromoter to study biologicals with ~200 nm-excited UVRR and on refurbishing a picosecond Nd:YAG laser so that it can directly probe picosecond dynamics of model peptides and organic molecules.
Current location: postdoctoral researcher in the Frontiera lab at the University of Minnesota.
B.S., Molecular Biology/Biochemistry, University of Pittsburgh, 2009
Ph.D., Molecular Biophysics & Structural Biology, University of Pittsburgh, 2016
- Levine AB, Punihaole D, Levine TB (2012) Characterization of the Role of Nitric Oxide and Its Clinical Applications. Cardiology 2012;122:55-68
- Xiong, K., D. Punihaole, & Asher, S.A. (2012). "UV Resonance Raman Spectroscopy Monitors Polyglutamine Backbone and Side Chain Hydrogen Bonding and Fibrillization." Biochemistry 51(29): 5822-5830.
- Cai Z, Zhang JT, Xue F, Hong Z, Punihaole D, Asher SA. 2D photonic crystal protein hydrogel coulometer for sensing serum albumin ligand binding. Anal Chem. 2014; 86(10):4840-7
- Punihaole, D.; Jakubek, R. S.; Dahlburg, E. M. ; Hong, Z.; Myshakina, N. S.; Geib, S.; Asher, S. A. UV Resonance Raman Investigation of the Aqueous Solvation Dependence of Primary Amide Vibrations. J. Phys. Chem. B. 119(10):3931-9
- Punihaole D, Hong Z, Jakubek RS, Dahlburg EM, Geib S, Asher SA. (2015) Glutamine and Asparagine Side Chain Hyperconjugation-Induced Structurally Sensitive Vibrations. J Phys Chem B. 119(41):13039-51
- Cai Z, Kwak DH, Punihaole D, Hong Z, Velankar SS, Liu X, Asher SA. (2015) A Photonic Crystal Protein Hydrogel Sensor for Candida albicans. Angew Chem Int Ed Engl. 54(44):13036-40
- Punihaole D, Workman RJ, Hong Z, Madura JD, Asher SA. (2016) Polyglutamine Fibrils: New Insights into Antiparallel β-Sheet Conformational Preference and Side Chain Structure. J Phys Chem B. 120(12):3012-26
- Sharma B, Cardinal MF, Ross MB, Zrimsek AB, Bykov SV, Punihaole D, Asher SA, Schatz GC, Van Duyne RP. (2016) Aluminum Film-Over-Nanosphere Substrates for Deep-UV Surface-Enhanced Resonance Raman Spectroscopy. Nano Lett. 2016 Dec 14;16(12):7968-7973.
- Punhaole D, Jakubek RS, Workman RJ, Marbella LE, Campbell P, Madura JD, Asher SA. (2017) Moomeric Polyglutamine Structures That Evolve into Fibrils. J Phys Chem B. 2017; 121(24): 5953-5967
- Punihaole D, Jakubek RS, Workman RJ, Asher SA. Interaction Enthalpy of Side Chain and Backbone Amides in Polyglutamine Solution Monomers and Fibrils. J Phys Chem Lett. 2018;9(8):1944-1950
Muwen Ben Kong
Thesis Title: Single-Molecule Studies of Rad4-Rad23 Reveal a Dynamic DNA Damage Recognition Process
Graduation: June 30, 2017
Our lab studies the nucleotide excision repair (NER) pathway in DNA repair. Ben's thesis project looked at the mechanism of DNA damage recognition by yeast NER protein Rad4-Rad23, using single-molecule techniques such as our DNA tightrope assay with quantum dot labeled proteins, as well as atomic force microscopy (AFM).
B.A. Physics, Knox College 2008
M.S. Physics, CMU 2010
PhD Advisor: Dr. Bennet Van Houten
Current Location: postdoc in Eric Greene lab, Columbia University (New York, NY) (per Oct 2017)
- Van Houten, B., and Kong, M. (2015) Eukaryotic Nucleotide Excision Repair, in Encyclopedia of Cell Biology, edited by Ralph A. Bradshaw and Philip D. Stahl, Academic Press.
- Kong M, Liu L, Chen X, Drisöhmcoll KI, Mao P, B S, Kad NM, Watkins SC, Bernstein KA, Wyrick JJ, Min JH, Van Houten B. Single-Molecule Imaging Reveals that Rad4 Employs a Dynamic DNA Damage Recognition Process. Mol Cell. 2016 Oct 20;64(2):376-387
- Kong M, Van Houten B. Rad4 recognition-at-a-distance: Physical basis of conformation-specific anomalous diffusion of DNA repair proteins. Prog Biophys Mol Biol. 2017 Aug;127:93-104
- Luo J, Kong M, Liu L, Samanta S, Van Houten B, Deiters A. Optical Control of DNA Helicase Function through Genetic Code Expansion. Chembiochem. 2017;18(5):466-469
- Kong M, Beckwitt EC, Springall L, Kad NM, Van Houten B. Single-Molecule Methods for Nucleotide Excision Repair: Building a System to Watch Repair in Real Time. Methods in Enzymology, DNA Repair Enzymes, Part B, Volume 592, Academic Press, 2017, 592: 213-257
- Beckwitt E, Kong M, Van Houten B. Studying Protein-DNA Interactions Using Atomic Force Microscopy. Semin Cell Dev Biol. 2017 Jun 30. pii: S1084-9521(17)30127-1
- Liu L, Kong M, Gassman NR, Freudenthal BD, Prasad R, Zhen S, Watkins SC, Wilson SH, Van Houten B. PARP1 changes from three-dimensional DNA damage searching to one-dimensional diffusion after auto-PARylation or in the presence of APE1. Nucleic Acids Res. 2017 45(22):12834-12847