This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. RG13 is a 637-aminoacid engineered allosteric molecular switch between beta-lactamase (BLA) and maltose binding protein (MBP) [Gutas G, Mitchell SF, Ostermeier M, (2004) Chem Biol 11:1483-1487;Guntas G, Mansell TJ, Kim JR, Ostermeier M, (2005) Proc Natl Acad Sci USA 102:11224-11229]. In the absence of maltose, MBP exists in an open form. Maltose binding is concomitant with a 35 degree bending motion about the hinge, resulting in the closed form of the protein [Sharff AJ, Rodseth LE, Spurlino JC, Quiocho FA, (1992) Biochemistry 31:10657-10663]. BLA is a monomeric enzyme that hydrolyzes the amide bond of the beta-lactam ring of beta-lactam antibiotic such as penicillin. The circularly permuted BLA was inserted into the MBP, and a switch (RG13) was identified in which its beta-lactam hydrolysis activity was compromised in the absence of maltose but increased 25-fold in the presence of maltose. We reasoned that in the switch the conformational change in the MBP domain upon maltose binding would propagates to the active site of the BLA domain and alter its catalytic properties, a mechanism analogous to natural allosteric effects. RG13 was identified from a combinational library rather than rationally designed, and thus, the molecular mechanism by which switching occurs is not known. Our goal is to develop a structural understanding of the switching mechanism in order to guide generation of further hypotheses such as optimizing and creating novel allosteric switch molecules for various applications. In order to achieve the goal, Dr. Ostermeier and co-workers are conducting NMR experiments including residual dipolar coupling (RDC), nuclear Overhauser enhancement (NOE), and paramagnetic relaxation enhancement (PRE) measurements. These measurements provide orientation and distance constraints for structural modeling RG13. We would first predict the structures of the switch using Rosetta NMR [Raman S, Lange OF, Rossi P, Tyka M, Wang X, et al. (2010) Science 327:1014-1018;Raman S, Huang YJ, Mao B, Rossi P, Aramini JM, et al. (2010) J Am Chem Soc 132:202-207;Shen Y, Vernon R, Baker D, Bax A, (2009) J Biomol NMR 43:63-78] and domain insertion [Berrando M, Ostermeier M, Gray JJ, (2008) Structure 16:513-527] approaches. Our previous NMR studies (15N, 1H-TROSY-HSQC) indicate that the individual domain structures of RG13 are substantially conserved from MBP and BLA [Wright CM, Majumdar A, Tolman JR, Ostermeier M, (2010) Proteins 78:1423-1430]. Therefore we can get good starting structures from Monte Carlo (MC) simulations with NMR constraints. Proteins, however, are flexible and dynamic. In order to achieve our goal to understand the allosteric switching mechanism of RG13, we have to understand conformational changes upon fusion of two parent proteins (BLA and MBP) and binding of maltose and their propagation from MBP to BLA through the flexible linkers. This dynamic process will be investigated by running molecular dynamics (MD) simulations. Structural model and its dynamics will be compared and tested with NMR relaxation data by running long time MD simulations (>60 ns) [Bhattacharya N, Yi M, Zhou HX, Logan TM, (2007) J Mol Biol 374:977-992]. We would model the structure in the presence of antibiotic molecules such as penicillin, which has not been parameterized for MD simulations. In order to prepare the parameters compatible to CHARMM force field format, we would perform quantum chemistry calculations with Gaussian03.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
3P41RR006009-20S1
Application #
8364341
Study Section
Special Emphasis Panel (ZRG1-BCMB-Q (40))
Project Start
2011-09-15
Project End
2013-07-31
Budget Start
2011-09-15
Budget End
2013-07-31
Support Year
20
Fiscal Year
2011
Total Cost
$1,036
Indirect Cost
Name
Carnegie-Mellon University
Department
Biostatistics & Other Math Sci
Type
Schools of Arts and Sciences
DUNS #
052184116
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Yonkunas, Michael; Buddhadev, Maiti; Flores Canales, Jose C et al. (2017) Configurational Preference of the Glutamate Receptor Ligand Binding Domain Dimers. Biophys J 112:2291-2300
Earley, Lauriel F; Powers, John M; Adachi, Kei et al. (2017) Adeno-associated Virus (AAV) Assembly-Activating Protein Is Not an Essential Requirement for Capsid Assembly of AAV Serotypes 4, 5, and 11. J Virol 91:
Subramanian, Sandeep; Chaparala, Srilakshmi; Avali, Viji et al. (2016) A pilot study on the prevalence of DNA palindromes in breast cancer genomes. BMC Med Genomics 9:73
Ramakrishnan, N; Tourdot, Richard W; Radhakrishnan, Ravi (2016) Thermodynamic free energy methods to investigate shape transitions in bilayer membranes. Int J Adv Eng Sci Appl Math 8:88-100
Zhang, Yimeng; Li, Xiong; Samonds, Jason M et al. (2016) Relating functional connectivity in V1 neural circuits and 3D natural scenes using Boltzmann machines. Vision Res 120:121-31
Lee, Wei-Chung Allen; Bonin, Vincent; Reed, Michael et al. (2016) Anatomy and function of an excitatory network in the visual cortex. Nature 532:370-4
Murty, Vishnu P; Calabro, Finnegan; Luna, Beatriz (2016) The role of experience in adolescent cognitive development: Integration of executive, memory, and mesolimbic systems. Neurosci Biobehav Rev 70:46-58
Lee, Tai Sing (2015) The visual system's internal model of the world. Proc IEEE Inst Electr Electron Eng 103:1359-1378
Kuhlman, Chris J; Anil Kumar, V S; Marathe, Madhav V et al. (2015) Inhibiting diffusion of complex contagions in social networks: theoretical and experimental results. Data Min Knowl Discov 29:423-465
Jurkowitz, Marianne S; Patel, Aalapi; Wu, Lai-Chu et al. (2015) The YhhN protein of Legionella pneumophila is a Lysoplasmalogenase. Biochim Biophys Acta 1848:742-51

Showing the most recent 10 out of 289 publications