Computation of Chemical Shift Tensors of Flavins and Their Responses to Non-Covalent Interactions and Covalent Modifications

This project makes use of Gaussian to perform ab-initio geometry optimization using density functional methods and a range of basis sets to obtain a computational model of the flavin geometry and its electronic structure. The optimized model is tested with respect to its ability to describe experimental situations by computation of predicted chemical shift tensor principal values which are then compared with measured chemical shift principal values. The benefits are two-fold: we are able to validate high-level calculations of flavin electronics that can then be used to understand the reactivity of flavins and the extent and ways in which this is changed by variations in the protein environment surrounding the flavin, and we are able to learn to interpret the observed chemical shift principal values in terms of specific interactions affecting the flavin and specific electronic consequences. The significance of this work is that flavins are the active ingredient in hundreds of enzymes fulfilling vital roles in biochemistry. They also have implications for integration in bioengineering of enzymes to conduct desired reactions , since proteins have very limited ability to conduct redox reactions whereas flavins are ideally suited to doing so in a tunable way. NMR chemical shifts are more sensitive to electronic perturbations in general than almost any other spectroscopic method, they can be detected in solid-state samples even for enzymes that are resistant to crystallisation and the chemical shifts of 13C and 15N atoms built right into the flavin ring reveal a large range of values. We have published the first solid-state NMR spectra of flavins and provided the first demonstration that the chemical shift principal values of the redox-active N atom changes by readily detectable amounts in response to formation of hydrogen bonds, even to remote protions of the flavin moiety. Moreover our computations (using Gaussian 03 on the DLX) were able to reproduce the experimentally observed changes providing the first validation of the capability of density functional calculations to describe and explain the consequences of these interactions which are fundamentally non-covalent and can be difficult to describe accurately. We are now undertaking more complicated systems, that provide a more realistic description of enzyme active sites. Our current target is the flavin site of flavodoxin from Desulfovibrio vulgarise. This is a heavily-studied system for which many crystal structures are available including the structures of mutant proteins. Most importantly there are published reduction midpoint potentials for these. Thus we are working towards calculations of the energies associated with electron acquisition in a computational model of the flavodoxin active site. These energies place a thermodynamic boundaries of the chemistry the bound flavin will be able to perform. Thus protein engineering efforts seek to modify these energies. We will learn from our studies whether computations can correctly predict the redox potentials of mutant flavodoxins relative to the wild-type. If so, then computations will be able to provide guidance to engineering efforts by allowing computational screening of numerous potential mutants so that only the most promising need be constructed. The computations will be validated by also computing chemical shifts for mutants deemed interesting, and NMR will be used to measure the chemical shifts for them and to test the computations' abilitiest to correctly describe the flavin electronics. If the computations are successful then we will be able to use them to understand the electronic mechanisms underlying changes in redox potentials produced by the mutations. This will pave the way to genuinely rational engineering of flavin redox potentials. To improve the computations and address any deficiencies in reproducing the midpoint potential changes, or the changes in chemical shifts producted by mutations, we will explore more comprehensive models in which the quantum description of the active site is embedded in a molecular mechanics model of the entire protein. Such hybrid QM-MM computations are by now routine in the field, but we have yet to accomplish one ourselves, so this is an important goal for the upcoming year. We are flexible as to which software to use, but need to have an MM package that interfaces readily with Gaussian, since may DFT engines used in QMMM do not calculate chemical shift tensors. Car-Parrinello has been recommended, as have AMBER and ONETEP.
Dr. Michael Sheetz has been an enormous help, but is clearly every bit as busy as the research faculty.

Software:

Gaussian (I would appreciate Gaussview compatible with a Mac interface or the Darwin Unix shell). Gaussian is already on the DLX and we have access to Gaussview for PCs. I am not sure however how up to date we are on our versions at UK.
AMBER and ONETEP are available on DLX but we have not learned how to run them from our terminals.
Car-Parrinello is on my wish list, we have not tried it. It is available from NWChem.

Students:

Warintra Pitsawong, Graduate, Chemistry

Collaborations:

Rajiv Kumar Kar, Technische Universitet of Berlin, Germany

Dr. Michael Sheetz: CCS, UK

Publications:

Publications based on work on DLX (Other papers date to the older supercomputers now retired)
D. Cui, R. L. Koder, Jr. P. L. Dutton and A.-F. Miller (2011) " 15N Solid-State NMR as a probe of Flavin H-bonding." J. Phys. Chem -B. 115 (24): 7788-7798. doi.org/10.1021/jp202138d

SDX Supercomputer Publications:

2014

1.Rathnayake, S., Unrine, J. M., Judy, J., Miller, A.-F., Rao, W., Bertsch, P. M. (2014) "A multi-technique investigation of phosphate induced transformation of ZnO nanoparticles." Environmental Science & Technology 48(9): 4757-4764. http://pubs.acs.org.ezproxy.uky.edu/doi/pdf/10.1021/es404544w(external link)
2.Sheng, Y., Abreu, I. A., Cabelli, D. E., Maroney, M. J., Miller, A.-F., Teixeira, M. and Valentine, J. S. (2014) "Superoxide dismutases and superoxide reductases" (2014) Chemical Reviews 2014 114:3854-3918.
3.Pitsawong, W., Hoben, J. P., Miller, A.-F. (2014) "Understanding the Broad Substrate Repertoire of Nitroreductase Based on its Kinetic Mechanism" the Journal of Biological Chemistry. 289:15203-15214.
4.Raghavendra M.P., Koteshwar, R., Miller, A., Lokanatha-Rai, K. M. "Antifungal activity of novel alkaloid 1HImidazole-4-carboxylic acid 2-ethyl-hexyl ester isolated from leaves of Prosopis juliflora (Sw.) DC." Submitted to Current Microbiology.

2013

Mattei, A., Mei, X., Miller, A.-F., Li, T. (2013) "Two Major Pre-Nucleation Species that are Conformationally Distinct and in Equilibrium of Self-Association" Crystal Growth & Design 13(8): 3303-3307. http://pubs.acs.org/doi/pdf/10.1021/cg401026j(external link)
Jackson, T. A., Gutman, C. T. , Maliekal, J., Miller, A.-F. Brunold, T. C. (2013) "Geometric and Electronic Structures of Manganese-substituted Iron Superoxide Dismutase" Inorganic Chemistry 52(6):3356-3367. http://pubs.acs.org/doi/full/10.1021/ic302867y(external link)
A.-F. Miller (2013) "Solid state NMR of flavins and flavoproteins" in press "Flavins and flavoprotein protocols" Schleicher, E. and Weber, S., Eds. (Elsevier).
A.-F. Miller (2013) "Superoxide Dismutases" Encyclopedia of Biophysics, G. Roberts, V. Davidson, Ed. pp 2517-2522.
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2012

T. Maly, D. Cui, R. G. Griffin and A.-F. Miller (2012) "Dynamic nuclear polarization based on an endogenous radical" J. Phys. Chem. -B 116: 7055-7065.
A.-F. Miller (2012) "Superoxide dismutases: ancient enzymes and new insights." FEBS Lett. 586: 585-595. http://www.sciencedirect.com/science/article/pii/S0014579311008271(external link)
A. A. Adeneye, P. A. Crooks, A. F. Miller, J. Goodman, O. O. Adeyemi, E. O. Agbaje (2012) "Isolation and structure elucidation of a new indole alkaloid, erinidine, from Hunteria umbellata seed." Pharmacologia 3(7): 204-214. DOI:10.5567/pharmacologia.2012.204.214.
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2011

B. Purushothaman, M. Bruzek, S. R. Parkin, A.-F. Miller and J. E. Anthony (2011) "Synthesis and structural characterization of crystalline nonacenes", VIP paper, Angewandte Chemie Int. Ed. 50(31) Cover pg. 6932 text pages 7013-7017 DOI : 10.1002/anie.201102671.
D. Cui, R. L. Koder, Jr. P. L. Dutton and A.-F. Miller (2011) " 15N Solid-State NMR as a probe of Flavin H-bonding." J. Phys. Chem -B. 115 (24): 7788-7798. doi.org/10.1021/jp202138d
N. B. Surmeli, N. K. Litterman, A.-F. Miller and J. T. Groves (2011) "Peroxynitrite Mediates Active Site Tyrosine Nitration in Manganese Superoxide Dismutase. Evidence of a Role for the Carbonate Radical Anion." J. Am. Chem. Soc. 132 (48), 17174–17185 http://pubs.acs.org/doi/abs/10.1021/ja105684w(external link) DOI: 10.1021/ja105684w
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2010

J. Johnston, H. Hassan, A.-F. Miller and M. Apicella (2010) "Sialic acid transport and catabolism are cooperatively regulated by SiaR and CRP in nontypeable Haemophilus influenzae" BMC Microbiology 10:240. DOI 10.1186 http://www.biomedcentral.com/1471-2180/10/240(external link)
T. Maly, A.-F. Miller and R. G. Griffin (2010) "In-situ High-Field Dynamic Nuclear Polarization : Direct and Indirect Polarization of 13C nuclei" Chem. Phys. Chem. 11(5): 999-1001. DOI 10.1002
A.-F. Miller, E. Yikilmaz and S. Vathyam (2010) " 15N-NMR characterization of His residues in and around the active site of FeSOD." Biochim. Biophys. Acta 1804: 275-284. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B73DJ-4XR5N6F-2&_user=16764&_coverDate=02%2F28%2F2010&_rdoc=5&_fmt=high&_orig=browse&_srch=doc-info(%23toc%2311472%232010%23981959997%231619106%23FLA%23display%23Volume)&_(external link) doi:10.1016/j.bbapap.2009.11.009
G. Osei-Prempeh, H.-J. Lehmler, A.-F. Miller, B. L. Knutson and S. E. Rankin (2010) "Fluorocarbon and hydrocarbon functional group incorporation into nanoporous silica employing fluorinated and hydrocarbon surfactants as templates" Microporous & Mesoporous Materials 129: 189-199.
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2008

A.-F. Miller (2008) "The shortest wire" Proc. Natl. Acad. Sci. U.S.A. 105 (21): 7341-7342. http://www.pnas.org/content/105/21/7341.full(external link)
L. E. Grove, J. Xie, E. Yikilmaz, A. Karapetyan, A.-F. Miller, and T. C. Brunold (2008) “Spectroscopic and Computational Insights into Second-Sphere Amino-Acid Tuning of Substrate Analogue/Active-Site Interactions in Iron(III) Superoxide Dismutase” Inorg. Chem. 47: 3993-4004. http://pubs.acs.org/cgi-bin/article.cgi/inocaj/2008/47/i10/pdf/ic702414m.pdf(external link) , DOI: 10.1021/ic702414m
L. Grove, J. Xie, E. Yikilmaz, A.-F. Miller, and T. C. Brunold (2008) "Spectroscopic and Computational Insights into Second-Sphere Contributions to Redox Tuning in Escherichia coli Iron Superoxide Dismutase" Inorg. Chem. 47: 3978-3992. http://pubs.acs.org/cgi-bin/article.cgi/inocaj/2008/47/i10/pdf/ic702412y.pdf(external link) , DOI: 10.1021/ic702412y
J. F. Cerda, R. L. Koder, B. R. Lichtenstein, C. M. Moser, A.-F. Miller, and P. L. Dutton (2008) "Hydrogen Bond-Free Flavin Redox Properties: Managing Flavins in Extreme Aprotic Solvents" Org. Biomol. Chem., 6: 2204-2212. DOI: 10.1039/ B801952E
A.-F. Miller (2008) "Redox tuning over almost 1 V in a structurally-conserved active site: lessons from superoxide dismutase.", Acc. Chem. Res. 41 (4) 501-510. http://pubs.acs.org/cgi-bin/article.cgi/achre4/2008/41/i04/pdf/ar700192d.pdf(external link) DOI: 10.1021/ar700237u
W. L. Boatright, M.S. Jahan, B.M. Walters, A.F. Miller, D. Cui, E.J. Hustedt and Q. Lei (2008) "Carbon Centered Radicals in Isolated Soy Proteins", J Food Science, 73(3):C222-226. doi: 10.1111/j.1750-3841.2008.00693.x

Grants

Miller, Anne Frances NO ID Nitroreductase, a basis for breadth Multiple Industry Sponsors 9/1/2014 - 1/31/2015 SCOPE

Most recent funding to this project: 1/Sep./06-31/Sep/10: Petroleum Research Foundation, 44321-AC4 $80,000 Direct (P.I.) ‘Solid-state NMR as a probe of flavin interactions, electronics and reactivity’.

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