Evgenii Kovrigin

Evgenii Kovrigin*, Ph.D.
Assistant Professor
phone: 414-288-7859
email: evgueni.kovriguine@marquette.edu

*spelling on official documents: "Evgueni Kovriguine"

Website: http://lineshapekin.net/


Dr. Evgenii Kovrigin obtained his doctoral degree in Chemistry from the Engelhardt Institute of Molecular Biology (Moscow, Russia) in 1999. His doctoral studies of protein thermodynamics in mixed solvents at the Institute of Protein Research (Pushchino, Russia) brought him "Microcal Young Scientist Award 1999" presented to him at 2nd International Conference on Applications of Biocalorimetry 1999 in Halle/Saale, Germany. Evgenii's post-doctoral work at UT Southwestern (Dallas, Texas) and Yale University (New Haven, Connecticut) was focused on determination of structure and dynamics of proteins using NMR spectroscopy. From 2006 to 2011, Evgenii was a faculty member at the Biochemistry Department of the Medical College of Wisconsin where he focused on dynamics and interactions of proteins involved in cancer signaling. He joined the Chemistry Department of Marquette University in 2011 to continue his studies of protein structure and dynamics.

Research Profile

Proteins are fascinating molecular machines central to cellular function. My laboratory aims to understand fundamental relationships between protein structure, dynamics, and interactions with ligands. We investigate protein interactions and dynamics from various viewpoints using Nuclear Magnetic Resonance (NMR) spectroscopy as a main tool complemented by a range of biophysical and biochemical methods. Our major research directions are described below:

Allosteric coupling in Ras GTPase

Ras is a small monomeric G protein and the "founding" member of the Ras superfamily. These proteins act as molecular switches responsible for signal transduction and regulation of a multitude of processes in a cell. Using NMR spin relaxation experiments, we discovered global conformational dynamics in Ras structure [14] that encompasses entire G domain from effector interface to the membrane-facing region of the protein molecule. Analysis of NMR titrations of Ras with small divalent ions revealed thermodynamic coupling of the guanine nucleotide pocket to a recently reported ion-binding site near the C-terminal region of Ras [22]. These findings suggest that the effector interface of Ras G domain is allosterically coupled to the membrane-bound C-terminal region. Such coupling may constitute a long-sought-for link between activation status of the G domain and its membrane interactions. Our current research aims to identify specific elements of Ras structure controlling this allosteric coupling mechanism.



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Ras dimerization state and lipid interactions

Ras is a recognized high-profile cancer drug target with NIH Ras Initiative investing ca. $10M a year to find inhibitors against Ras. Dimerization of Ras molecules at the membrane has been recently hypothesized as a new mechanism of Ras regulation possibly amenable to inhibition by small molecules. To establish whether and how Ras may self-associate, we investigated possible direct interactions between G domains in a tethered construct (effectively, creating a high local concentration of Ras). Measurements of polarization anisotropy and NMR chemical shift perturbations ruled out biologically relevant self-association of G domains. We concluded that Ras dimerization (if it occurs at all) cannot be driven by protein-protein interactions; instead, must be mediated by a "third party" - lipids of plasma membrane or some protein effector [27].


Our current efforts are focused on detailing a unique property of Ras as a membrane protein: its ability to change its membrane nanodomain localization upon activation (loading with GTP). For example, H-Ras in its inactive state was reported to be uniformly distributed in cellular membranes while being excluded from lipid rafts upon activation. To resolve details of this self-sorting mechanism, we created semisynthetic lipidated Ras samples in model raft-containing membranes. Using FRET between protein and lipid domain markers detected through time-resolved fluorescence spectroscopy, we investigated lipid domain preferences for a range of Ras constructs in active and inactive state. Future steps in this project will include all four Ras isoforms in asymmetric lipid bilayers closely mimicking the domain structure of the inner leaflet of the plasma membrane.

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NMR of supramolecular protein-lipid complexes

Cytochrome P450 oxidoreductase (POR) acts in the electron transfer chain in liver and other tissues. A product of a single gene in human tissues, POR supplies electrons to 48 cytochromes P450 enabling synthesis of steroids and prostaglandins as well as deactivation of a majority of therapeutic drugs. POR undergoes large conformational changes in its functional cycle and requires membrane attachment for productive interactions with cytochromes P450. To date, association with membrane surface and dynamic nature of POR prevented resolution of the structural features of its complex with P450.

In our work, we aim to visualize functional conformational transitions in POR and resolve structural and kinetic aspects of POR-P450 interactions using methyl-TROSY NMR spectroscopy. To enable stable membrane attachment and uniformly monomeric state of POR, we are preparing samples incorporated in lipid nanodiscs. Considering large molecular weight of the POR-P450-Nanodisc complex, we rely on sensitivity of methyl-TROSY to follow the protein-protein interactions and conformational changes throughout the entire functional cycle of the POR-P450 system.

por1 porp450nd

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NMR line shape analysis

NMR spectroscopy provides a unique window into kinetic mechanisms of protein-ligand interactions. Proteins rarely (if ever) bind their ligands following a two-state, Emil Fischer's "lock-and-key" mechanism. Instead, they often transition between different conformations to accommodate the ligand. Line shape of the NMR signal reflects multiple steps in a binding reaction, which may be identified using NMR line shape analysis [19]. In a series of collaborative studies, we demonstrated practical utility of NMR line shape fitting to extract kinetic and thermodynamic parameters of protein interactions [15, 16, 21, 24, 31]. Our most recent work focused on chitosanase enzyme binding its substrate, oligomeric chitosan. Three-state fitting identified the induced fit as the most likely mechanism of the enzyme-substrate interaction. We emphasized that NMR line shape patterns may be highly ambiguous - always requiring consideration of a range of alternative mechanisms [31].

simulated line shapes  structure of chitosanase

Despite tremendous information content of the NMR line shape, the method did not become mainstream in protein NMR yet becase of its relatively complicated formalism. We are developing software for semi-automated simulation and fitting of line shapes, LineShapeKin Simulation, and the NMR line shape module for the Integrative Data Analysis Platform (IDAP) to help make this technique accessible to a broader NMR community.

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Global analysis of ligand-binding data

Detailed resolution of protein-ligand interaction mechanisms is crucial for understanding biological function and development of new pharmaceutical drugs. Ligand binding events are often studied by a number of different biophysical approaches illuminating the interaction mechanisms from complementary perspectives. Complex interaction modes require multi-state thermodynamic and kinetic models that are overparameterized when fit to datasets of just one type. Including data of different types in one fitting session allows for simultaneous optimization of thermodynamic and kinetic parameters with respect to all data - making it possible to resolve accurate details of a complex biomolecular system.


To enable global analysis of datasets originating from different types of measurements, we are developing the Integrative Data Analysis Platform (IDAP) software based on object-oriented MATLAB code. The key feature of IDAP algorithm is its ability to peform simultaneous optimization of parameters in datasets of very different types yet sharing a global thermodynamic and kinetic model. Such datasets may originate from experimental interrogation of the same protein-ligand interaction using, for example, relaxation dispersion NMR experiments, Isothermal Titration Calorimetry, NMR line shape analysis, rapid mixing kinetics, ultrafast transient absorption spectroscopy, etc.

In IDAP, each dataset type is a data class that "knows" its own specific mathematical formalism to describe experimental signals. At the same time, all datasets rely on one chosen global thermodynamic and kinetic model. The models may be easily switched enabling extensive hypothesis testing. The IDAP algorithm allows for a very flexible way of linking parameters across multiple datasets: a user may make parameters completely local (dataset-specific), global for all datasets or linked to any intermediate degree (for example, among selected sets of data).

IDAP modules that enable analysis of data from different biophysical techniques were successfully utilized in a number of biophysical studies:

We continue development of the IDAP to enable analysis of data from a range of additional biophysical techniques, and welcome new collaborations.

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Development of a new FRET method based on ultrafast transient absorption spectroscopy

Distances between selected sites in proteins and protein complexes are often measured using Forster Resonance Energy Transfer (FRET). This becomes difficult to do when proteins have intrinsic fluorophores that would interfere with FRET between donors and aceptors. Cytochrome P450 oxidoreductase (POR) harbors flavin cofactors with absorption bands extending from ultraviolet to green wavelengths. Red-Infrared donor-acceptor pairs are the only choice for FRET in POR, and most of them have short fluorescence life times requiring very accurate time-resolved detection. We are developing an application of ultrafast transient absorption spectroscopy to quantify FRET in such demanding systems in collaboration with laboratory of Dr. Jier Huang at Marquette Chemistry. Application of this novel FRET method to a cytosolic domain of POR allowed to assess conformational states of this dynamic protein in oxidized and reduced states [29]. We continue development of this approach to extend it to the membrane-bound POR interacting with cytochrome P450.


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New tools for fluorescence spectroscopy

Two dimensional fluorescence detection

Complex biochemical samples often include multiple fluorophores such as aromatic aminoacids, metabolites and specific fluorescent labels.Two-dimensional fluorescence measurements were established as a preferred method for analysis of such samples in which the emission spectra are collected for all excitation wavelengths. The resulting two-dimensional spectrum enables identification of multiple fluorophores in the mixture as well as evolution of their concentrations - even in the presence of significant spectral overlap. To facilitate acquisition and analysis of such two-dimensional fluorescence data, we developed the Fluorescence2D  software particularly designed for use with the accelerated “triangular” acquisition schemes [26].

Fluorescence2D is freely available for academic laboratories.



Novel methods for fluorescence measurements of supported lipid bilayers

Supported phospholipid bilayers are a convenient model of planar lipid membranes, useful for studies of integral and peripheral membrane proteins. Typically, these bilayers are created on a horizontal glass surface and observed using confocal microscopes. However, spectral recording or time-resolved studies of supported bilayers are not generally possible unless one has a state-of-the-art confocal microscope with spectral and/or time resolution. We developed a simple method that allows recording of fluorescence of a supported lipid bilayer in a conventional horizontal-beam spectrofluorometer equipped with standard monochromators and/or time-resolved capability [28].

In our method, the supported bilayers are formed on a surface of a standard fluorometer cell and observed in a conventional spectrofluorometer using a specially designed cell adaptor (patent pending). Since no modifications to the spectrofluorometer are required, a range of experiments with membrane proteins in supported bilayers become possible without a need for investment in high-end confocal microscopes. We continue development of this approach to enable new analytical and diagnostic methods particularly for low-budget settings such as college and public health department labs.



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31. Shinya S, Ghinet MG, Brzezinski R, Furuita K, Kojima C, Sneha Shah, Kovrigin EL*, Fukamizo T. (2017)  NMR Line Shape Analysis of a Multi-State Ligand Binding Mechanism in Chitosanase,  Journal of Biomolecular NMR, 67, 309-319
*) joint corresponding author.

30. Yang S, Pattengale B, Kovrigin EL, Huang J (2017) Photoactive Zeolitic Imidazolate Framework as Intrinsic Heterogeneous Catalysts for Light-Driven Hydrogen Generation, ACS Energy Letters, 2, 75-80

29. Kovrigina EA, Pattengale B, Xia C, Galiakhmetov AR, Huang J, Kim J-JP, Kovrigin EL, Conformational states of cytochrome P450 oxidoreductase evaluated by FRET using ultrafast transient absorption spectroscopy, (2016) Biochemistry, 55, 5973

28. Kovrigina EA, Kovrigin EL, Fluorescence of supported phospholipid bilayers recorded in a conventional horizontal-beam spectrofluorometer, (2015) Journal of Fluorescence, 26, 379-383

27. Kovrigina EA, Galiakhmetov AR, Kovrigin EL (2015) Ras G domain lacks intrinsic propensity to form dimers, Biophysical Journal 109, 1000-1008

26. Kovrigin EL (2014) Fluorescence2D: Software for Accelerated Acquisition and Analysis of Two-Dimensional Fluorescence Spectra, PLoS One 9, e101227

25. Li T, Simonds L, Kovrigin EL*, Noel D (2014) In vitro Biosynthesis and Chemical Identification of UDP-N-acetyl-D-quinovosamine (UDP-QuiNAc), Journal of Biological Chemistry 289, 18110-20
*) joint corresponding author.

24. Hawse WF, De S, Greenwood AI, Nicholson LK, Zajicek J, Kovrigin EL, Kranz DM, Garcia KC, and Baker BM, (2014) TCR Scanning of Peptide/MHC through Complementary Matching of Receptor and Ligand Molecular Flexibility, Journal of Immunology 192, 2885-2891

23. Whitney DS, Peterson FC, Kovrigin EL, Volkman BF,  (2013) Allosteric activation of the Par-6 PDZ via a partial unfolding transition, Journal of the American Chemical Society, 135, 9377-9383

22. O’Connor C, Kovrigin EL (2012) Characterization of the second ion-binding site in the G domain of H-Ras,  Biochemistry, 51, 9638-9646

21. Gagné D, Charest L-A, Kovrigin EL, and Doucet N, Conservation of flexible residue clusters among structural and functional enzyme homologues (2012) Journal of Biological Chemistry, 287, 44289-44300

20. De S, Greenwood AI, Rogals M, Kovrigin EL, Lu KP, and Nicholson LK, (2012) Complete Thermodynamic and Kinetic Characterization of the Isomer-Specific Interaction between Pin1-WW Domain and the Amyloid Precursor Protein Cytoplasmic Tail Phosphorylated at Thr66, Biochemistry, 51, 8583-8596

19. Kovrigin EL (2012) NMR line shapes and multi-state binding equilibria, Journal of Biomolecular NMR, 53, 257-270

18. Buhrman G., O'Connor C, Zerbe B, Kearney BM, Napoleon R, Kovrigina EA, Vajda S, Kozakov D, Kovrigin EL*, Mattos C, (2011) Analysis of binding site hot spots on the surface of Ras GTPase, Journal of Molecular Biology, Journal of Molecular Biology, 413, 773-789
*) joint corresponding author

17. O’Connor C, Kovrigin EL (2011)Assignments of Backbone 1H, 13C and 15N Resonances in H-Ras (1-166) Complexed with GppNHp at Physiological pH, Journal of Biomolecular NMR Assignment 6, 91-93

16. Greenwood AI, Rogals MJ, De S, Kovrigin EL and Nicholson LK (2011) Complete Determination of the Pin1 Catalytic Domain Thermodynamic Cycle by NMR Lineshape Analysis, Journal of Biomolecular NMR, Journal of Biomolecular NMR, 51, 21-34

15. Doucet N, Khirich G, Kovrigin EL, and Loria JP (2011)The alteration of hydrogen bonding in the vicinity of histidine 48 disrupts millisecond motions in RNase A, Biochemistry, 50, 1723-1730.

14. O'Connor C, Kovrigin EL (2008)Global conformational dynamics in Ras. Biochemistry, 47 (39), 10244-10246.
This publication was ranked the top "Most-Accessed Rapid Report in Biochemistry in 2008".

13. Watt, ED, Shimada, H, Kovrigin, EL, and Loria, JP (2007)"The mechanism of rate-limiting motions in enzyme function.", PNAS, 104, 11981-11986 (2007)
This publication was presented in Research Highlights. Nat. Struct. Mol. Biol., 14, 688.

12. Kovrigin EL and Loria JP (2006)"Characterization of the transition state of functional enzyme dynamics.", J. Am. Chem. Soc., 128:7724-7725

11. Kovrigin EL, Kempf JG, Grey MJ, Loria JP (2006)"Faithful Estimation of Dynamics Parameters from CPMG Relaxation Dispersion Measurements", J. Mag. Res., 180:93-104

10. Kovrigin EL, Loria J (2006)"Enzyme dynamics along the reaction coordinate: Critical role of a conserved residue", Biochemistry 45, 2636-2647

9. Kim Y, Kovrigin EL, Eletr Z (2006)"NMR studies of Escherichia coli acyl carrier protein: Dynamic and structural differences of the apo- and holo-forms", Biochem. Biophys. Res. Commun. 341: 776-783

8. Kovrigin EL, Cole R, Loria JP (2003)"Temperature dependence of the backbone dynamics of Ribonuclease A in the ground state and bound to the inhibitor, 5'-phosphothymidine (3'-5')-pyrophosphate adenosine 3'-phosphate."Biochemistry 42: 5279-5291

7. Chen X, Tomchick DR, Kovrigin E, Arac D, Machius M, Sudhof TC, Rizo J (2002)"Three-dimensional structure of the complexin/SNARE complex."Neuron 33: 397-409

6. Kovrigin E.L., Potekhin S.A.  (2000) On the stabilizing action of protein denaturants: acetonitrile effect on stability of lysozyme in aqueous solutions. Biophysical Chemistry 83, 45-59

5. Potekhin S.A., Kovrigin E.L. (1998) Influence of kinetic factors on heat denaturation and renaturation of biopolymers, Biofizika 43, 223-232  (in Russian)/ English translation published: Potekhin S.A., Kovrigin E.L. (1998) Effect of kinetic factors on heat denaturation and renaturation of biopolymers, Biophysics 43, 198-207

4. Potekhin S.A., Kovrigin E.L. (1998) Folding under inequilibrium conditions as a possible reason for partial irreversibilty of heat-denatured proteins: computer simulation study.  Biophysical Chemistry 73, 241-248

3. Kovrigin E.L., Potekhin S.A. (1997) Preferential solvation changes upon lysozyme heat denaturation in mixed solvents,  Biochemistry 36,  9195-9199.

2. Kovrigin E.L., Potekhin S.A. (1996)  A microcalorimetric study of dimethylsulfoxide effect on heat  denaturation of lysozyme, Biofizika  41, 1201-1206 (in Russian)/ English translation published: Kovrigin Ye.L., Potekhin S.A. (1996) Microcalorimetric study of the effect of dimethylsulphoxide on the heat denaturing of lysozyme, Biophysics 41, 1219-1224

1. Kovrigin E.L., Kirkitadze M.D., Potekhin S.A.  (1996) Enthalpy of stabilization of hen egg lysozyme structure in dimethylsulfoxide aqueous solutions, Biofizika 41, 549-553 (in Russian)/ English translation published: Kovrigin Ye.L., Kirkitadze M.D., Potekhin S.A. (1996) Enthalpy of stabilization of the structure of hen's egg lysozyme in aqueous solutions of dimethyl sulphoxide, Biophysics 41, 547-551..


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