a) Bioengineering of Gas Vesicles (GVs)
In my postdoctoral research, I led the project of developing acoustically modulated magnetic resonance imaging (AM-MRI), a new deep-tissue imaging modality. The work leverages the bioengineering of gas vesicles, a unique group of genetically encoded protein nanostructures that are dually responsive to ultrasound wave and magnetic field. Additionally, I am involved in projects including hyperpolarized xenon MRI, surface plasmon resonance imaging, and biochemical studies on gas vesicles.
- Lu GJ #, Farhadi A#, Mukherjee A, Shapiro MG (2018) Proteins, air and water: reporter genes for ultrasound and magnetic resonance imaging. Current Opinion in Chemical Biology 45, 57-63. (# co-authors)
- Lu GJ, Farhadi A, Szablowski JO, Lee-Gosselin A, Barnes SR, Lakshmanan A, Bourdeau RW, Shapiro MG (2018) Acoustically modulated magnetic resonance imaging of gas-filled protein nanostructures. Nature Materials (in press)
Gas vesicles (GVs) can produce robust susceptibility-based MRI contrast, and the contrast is “erasable” by ultrasound pulses, enabling the selective detection of GVs without confounding endogenous tissue contrast.
- Farhadi A, Ho G, Kunth M, Ling B, Lakshmanan A, Lu GJ, Bourdeau RW, Schröder L, Shapiro MG (2018) Recombinantly Expressed Gas Vesicles as Nanoscale Contrast Agents for Ultrasound and Hyperpolarized MRI. AIChE Journal (in press)
- Lakshmanan A #, Lu GJ #, Farhadi A #, Nety SP #, Kunth M, Lee-Gosselin A, Maresca D, Bourdeau RW, Yin M, Yan J, Witte C, Malounda D, Foster FS, Schröder L, Shapiro MG (2017) Preparation of biogenic gas vesicle nanostructures for use as contrast agents for ultrasound and MRI. Nature Protocols 12(10):2050-2080. (# co-authors)
A detailed protocol on the expression, purification, functionalization, ultrasound imaging and hyperpolarized xenon MRI of gas vesicles (GVs).
- Mukherjee A, Davis HC, Ramesh P, Lu GJ, Shapiro MG (2017) Biomolecular MRI reporters: Evolution of new mechanisms. Prog. Nucl. Magn. Reson. Spectrosc. 102-103:32-42.
- Maley AM, Lu GJ, Shapiro MG, Corn RM (2017) Characterizing Single Polymeric and Protein Nanoparticles with Surface Plasmon Resonance Imaging Measurements. ACS Nano 11(7):7447-7456.
- Barskiy DA, Coffey AM, Nikolaou P, Mikhaylov DM, Goodson BM, Branca RT, Lu GJ, Shapiro MG, Telkki V-V, Zhivonitko VV, Koptyug IV, Salnikov OG, Kovtunov KV, Bukhtiyarov VI, Rosen MS, Barlow MJ, Safavi S, Hall IP, Schröder L, Chekmenev EY (2017) NMR Hyperpolarization Techniques of Gases. Chemistry 23(4):725-751.
b) Membrane Protein Structural Biology
The general goal of my Ph.D. research was to develop a method that could determine atomic-resolution structures of membrane proteins in their native lipid bilayer environment. To achieve the goal, I explored novel methods in solid-state nuclear magnetic resonance (ssNMR). I pushed the technological frontier of the method by solving the structure of a challenging target at the time, the mercury transporter protein MerF. In addition to being a proof-of-concept for the methodology, the structure of MerF revealed large conformational rearrangement in response to terminal peptide truncation, which served as an example of the importance of studying full-length, unmodified membrane proteins.
- Radoicic J, Lu GJ, Opella SJ (2014) NMR structures of membrane proteins in phospholipid bilayers. Q. Rev. Biophys. 47(3):249-283.
- Tian Y, Lu GJ, Marassi FM, Opella SJ (2014) Structure of the membrane protein MerF, a bacterial mercury transporter, improved by the inclusion of chemical shift anisotropy constraints. J. Biomol. NMR 60(1):67-71.
- Lu GJ, Opella SJ (2014) Resonance assignments of a membrane protein in phospholipid bilayers by combining multiple strategies of oriented sample solid-state NMR. J. Biomol. NMR 58(1):69-81.
A summary of the strategy to combine multiple methods for resonance assignment, the key bottleneck of solving membrane protein structures by oriented sample solid-state NMR.
- Lu GJ, Tian Y, Vora N, Marassi FM, Opella SJ (2013) The structure of the mercury transporter MerF in phospholipid bilayers: a large conformational rearrangement results from N-terminal truncation. J. Am. Chem. Soc. 135(25):9299-9302.
The structure of the full-length mercury transporter protein MerF (PDB: 2M67) revealed ~ 90° conformational rearrangement of an α-helix compared to the structure of a truncated construct. The directions of the α-helix involved are highlighted by green arrows on the structures.
c) Method Development in Solid-state NMR
In addition to the biology projects, I pioneered several new ssNMR methodologies. Oriented Sample ssNMR (OS ssNMR) was a method to study membrane proteins reconstituted in lipid bilayers that could spontaneously align to the magnetic field. I targeted at two main bottlenecks of the methodology, a) resonance assignment and b) spectral resolution, and pioneered dipolar coupling correlated isotropic chemical shift (DCCICS) analysis and MSHOT-Pi4/Pi pulse sequence. In addition, I contributed to the developments of rotational alignment (RA) magic-angle-spinning (MAS) ssNMR, mismatched Hartmann−Hahn (MMHH) spin diffusion, and q-titration of lipid bicelle.
- Lu GJ, Park SH, Opella SJ (2012) Improved 1H amide resonance line narrowing in oriented sample solid-state NMR of membrane proteins in phospholipid bilayers. J. Magn. Reson. 220:54-61.
The initial discovery that MSHOT-Pi4/Pi pulse sequence improved 1H spectral resolution by > 2 folds, and the improvement pertained specifically to membrane protein samples.
- Das BB, Nothnagel HJ, Lu GJ, Son WS, Tian Y, Marassi FM, Opella SJ (2012) Structure determination of a membrane protein in proteoliposomes. J. Am. Chem. Soc. 134(4):2047-2056.
- Son WS, Park SH, Nothnagel HJ, Lu GJ, Wang Y, Zhang H, Cook GA, Howell SC, Opella SJ (2012) ‘q-Titration’ of long-chain and short-chain lipids differentiates between structured and mobile residues of membrane proteins studied in bicelles by solution NMR spectroscopy. J. Magn. Reson. 214(0):111-118.
- Lu GJ, Son WS, Opella SJ (2011) A general assignment method for oriented sample (OS) solid-state NMR of proteins based on the correlation of resonances through heteronuclear dipolar couplings in samples aligned parallel and perpendicular to the magnetic field. J. Magn. Reson. 209(2):195-206.
A new method, “dipolar coupling correlated isotropic chemical shift analysis” (DCCICS), for resonance assignment by oriented sample solid-state NMR.
- Marassi FM, Das BB, Lu GJ, Nothnagel HJ, Park SH, Son WS, Tian Y, Opella SJ (2011) Structure determination of membrane proteins in five easy pieces. Methods 55(4):363-369.
- Knox RW, Lu GJ, Opella SJ, Nevzorov AA (2010) A Resonance Assignment Method for Oriented-Sample Solid-State NMR of Proteins. J. Am. Chem. Soc. 132(24):8255-8257.
d) Theoretical Works on NMR Pulse Sequences
Driven by the finding that resolution enhancement of MSHOT-Pi4/Pi pulse sequence occurred selectively in membrane protein samples but not in microcrystalline samples, I analyzed the quantum mechanics theory underlying the pulse sequence and unveiled that MSHOT-Pi4/Pi sequence was resistant to the interference effects from the motion of membrane proteins. This “motion-adapted” feature was generalized to the design of other NMR pulse sequences and, as another example, I showed that the proton-relay mechanism played the major role in dilute spin exchange of oriented samples and how membrane protein motion could enable spin diffusion through forbidden transitions.
- Lu GJ, Opella SJ (2014) Mechanism of dilute-spin-exchange in solid-state NMR. J. Chem. Phys. 140(12):124201.
An experimental proof that the proton-relay mechanism played the central role in spin exchange in oriented sample solid-state NMR.
- Lu GJ, Opella SJ (2013) Motion-adapted pulse sequences for oriented sample (OS) solid-state NMR of biopolymers. J. Chem. Phys. 139(8):084203.
Numerical simulations to illustrate the underlying “motion-adapted” principle for the superior performance of MSHOT-Pi4/Pi pulse sequence on membrane protein samples.
e) Protein X-ray Crystallography
I conducted an undergraduate honors research project on the expression, purification, crystallization, and preliminary X-ray analysis of the arginine repressor protein ArgR from M. tuberculosis. My involvement in the project continued in the following year that resulted in the crystal structures of the C-terminal domain, the full-length protein, and the protein-DNA complex of ArgR.
- Cherney LT, Cherney MM, Garen CR, Lu GJ, James MNG (2008) Crystal Structure of the Arginine Repressor Protein in Complex with the DNA Operator from Mycobacterium tuberculosis. J. Mol. Biol. 384(5):1330-1340.
- Cherney LT, Cherney MM, Garen CR, Lu GJ, James MNG (2008) Structure of the C-terminal domain of the arginine repressor protein from Mycobacterium tuberculosis. Acta Crystallogr D Biol Crystallogr 64(Pt 9):950-956.
- Lu GJ, Garen CR, Cherney MM, Cherney LT, Lee C, James MNG (2007) Expression, purification and preliminary X-ray analysis of the C-terminal domain of an arginine repressor protein from Mycobacterium tuberculosis. Acta Crystallogr Sect F Struct Biol Cryst Commun 63(Pt 11):936-939.