Furey Lab

The Furey lab concentrates on the following:

1) Determination and analysis of structure-function relationships in macromolecules of biological interest, including thiamin diphosphate dependent enzymes, immunoglobulins, bacterial toxins, digestive enzymes and other proteins. X-Ray Crystallography is the primary method employed.

2) Development of techniques for the determination and analysis of macromolecular crystal structures.

3) Application /development of computing techniques and algorithms for understanding biological function on a molecular level.

 

Below displays the Research Details from the profile of each member of the lab.

Palaniappa Arjunan, PhD

Dr. Arjunan determines the structure of macromolecules of biological interest, and then analyses structure-function relationships. He primarily uses X-ray crystallography to accomplish this.

 

Dr. Arjunan's current research includes the high resolution three-dimensional structure determination of thiamin diphosphate(ThDP)-dependent enzymes, the yeast pyruvate decarboxylase (PDC) and pyruvate dehydrogenase multienzyme complex (PDHc) from Escherichia coli. The refined structure is then used to address long-standing issues regarding the structure and function of thiamin diphosphate-dependent enzymes. The structure determination also includes the structure of PDHc E1 in complex with a covalently bound reaction intermediate analogue. Other interests are: a) the crystal structural analysis of native and mutant ThDP-dependent enzymes, either alone or in complexes with substrates, inhibitors, activators or with other related enzymes and b) development of techniques for the determination and analysis of macromolecular crystal structure.


William Furey, PhD

Dr. Furey’s research involves the structure determination and analysis of large biological molecules and complexes by x-ray crystallography, and correlating the results with known functions. The work currently focuses on thiamin (vitamin B1) dependent enzymes and cell cycle regulating enzymes, as well as crystallographic methods development. Results of these studies could lead to development of therapeutic agents directed against pathogenic organisms, and anti-cancer drugs.

 

The pyruvate dehydrogenase multienzyme complex (PDHc, MW 4.7 million Daltons, 60 protein subunits & 60 active sites for the E. coli version) is present in most organisms and is critical for carbohydrate metabolism where it converts pyruvate, the product of glycolysis, to acetyl-CoA via a complicated process of substrate channeling within the confines of the complex. Structural analyses of the complex and its three major enzymatic components E1 (24 copies), E2 (24 copies), & E3 (12 copies) are underway, and Dr. Furey has already determined high resolution crystal structures for some of the components and reaction intermediates from the E. coli version.

 

The E1 components are rate determining and require thiamin diphosphate as a cofactor, but must interact with a flexible segment [lipoyl domain (LD) and associated lipoamide side chain] on an E2 to transfer the first reaction product, an acetyl group, to the E2 active site. The acetyl group is then transferred to co-enzyme A within the E2 active site, and the product acetyl-CoA is released. The E2 bound lipoamide group (now reduced) then moves to an E3 (FAD dependent) active site, where it is oxidized to restore the initial conditions. Binding of the flexible segment to E1 and E3 subunits is mediated by additional binding to a peripheral subunit-binding domain (PSBD), shown bound to E1.

 

 

Mechanistic details regarding the catalytic reactions in each active site are sought, as well as identifying structural aspects critical for assembly of the individual components to form the complete multienzyme complex. Specific mutations in some of the components are associated with hereditary diseases in humans, and detailed analyses of the structure-function relationships may suggest development of plausible therapeutic agents to counter the effects of the mutations. Additionally, given the critical nature of this system in overall energy production for cellular function, development of inhibitors binding at any of the catalytic sites, or at sites disrupting protein-protein assembly, may considerably weaken or kill the organism. Lack of appreciable sequence homology between PDHc’s from humans and pathogenic bacteria therefore suggests that effective, pathogen specific antibacterial agents may be developed.

 

Early expression or over expression of Cdc25 proteins can cause the cell to prematurely progress leading to oncogenic effects, making these enzymes exciting targets for anti-cancer drug development. As part of a collaborative effort with Dr. John Lazo’s group, several potent inhibitors of Cdc25 proteins have been discovered, and structural analysis of their complexes with the enzymes are underway to reveal both where and how these inhibitors function. Dr. Furey has crystallized the catalytic domain of Cdc25b and determined its high-resolution structure. His group is currently co-crystallizing the catalytic domain with several inhibitors, as a step towards development of effective anti-cancer agents via structure-based drug design procedures.

 

In collaboration with the Hauptman-Woodward Institute for Medical Research, Dr. Furey's group is developing new computational methods for solving macromolecular crystal structures by automated techniques. This work involves creating and developing a software package BnP, which is a merging of the PHASES package developed in the Furey lab, and the SnB package developed in Buffalo. A simple, graphical user interface is developed to enable automatic creation of an interpretable electron density map starting from observed x-ray diffraction data, with only a few mouse clicks and text field entries required. This will invoke automatic scaling of data, determination of heavy atom/anomalous scatterer sites, refinement and validation of sites, calculation of protein phases, phase refinement, and phase improvement via solvent flattening/negative density truncation. A few more mouse clicks enable automated building of a complete or nearly complete model by interfacing with other externally developed software. The idea is to make it simple for novices to determine good quality crystal structures, while enhancing the productivity of more sophisticated users as well.


Matthew J. Whitley, PhD

Dr. Whitley’s research is focused in the world of integrative structural biology/pharmacology.  He makes use of many tools in the field, chief among them X-ray crystallography, solution nuclear magnetic resonance spectroscopy, and small-angle X-ray scattering, to study proteins and protein complexes of biomedical relevance.  Currently, he is focused on understanding structure-function relationships in the pyruvate dehydrogenase multienzyme complex, a key metabolic complex that links glycolysis and the citric acid cycle.  The bacterial version of this complex is of interest as an antibiotic target, while malfunction of the human version has severe neurological consequences during development and after birth.  Other previous and ongoing work includes structural and biophysical characterization of mutants of gammaD-crystallin, which is associated with cataract formation in humans and many other species.

Palaniappa Arjunan, PhD
Research Instructor


Matthew J. Whitley, PhD
Research Instructor

Palaniappa Arjunan, PhD

Journal Articles

Nemeria N, P Arjunan, K Chandrasekhar, M Mossad, K Tittmann, W Furey and F Jordan.  Communication between thiamin cofactors in the Escherichia coli pyruvate dehydrogenase complex E1 component active centers.  J Biol Chem 285:11197-11209, 2010.
Jordan F, P Arjunan, S Kale, N Nemeria and W Furey.  Multiple roles of mobile active center loops in the E1 component of the Escherichia coli pyruvate dehydrogenase complex:  Linkage of protein dynamics to catalysis.  J Mol Catal B:  Enzym 61:14-22, 2009.
Kale S, P Arjunan, W Furey and F Jorda. A dynamic loop at the active center of the Escherichia coli pyruvate dehydrogenase complex E1 component modulates substrate utilization and chemical communication with the E2 component, n. J Biol Chem 282:28106-28116, 2007.
Arjunan P, M Sax, A Brunskill, K Chandrasekhar, N Nemeria, S Zhang, F Jordan and W. Furey. A thiamin-bound, pre-decarboxylation reaction intermediate analogue in the pyruvate dehydrogenase E1 subunit induces large-scale disorder-to-order transformations in the enzyme and reveals novel structural features in the covalently bound adduct. J Biol Chem 281:15296-15303, 2006.
Nemeria N, K Tittmann, E Joseph. L Zhou, MB Vazquez-coll, P Arjunan, G Hubner, W Furey and F Jordan. Glutamate 636 of the Escherichia Coli Pyruvate dehydrogenase-E1 participates in active center communication and behaves as an engineered acetolactate synthase with unusual stereoselectivity. J Biol Chem 280:21473-21482, 2005.
Arjunan P, K Chandrasekhar, M Sax, A Brunskill, N Nemeria, F Jordan and W Furey. Structural determinants of enzyme binding affinity: The E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate. Biochemistry 43:2405-2411, 2004.
Jordan F, N Nemeria, Y Yan, S Zhang, P Arjunan and W Furey. Presence of the 1,4’-imino tautomer of thiamin diphosphate on the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex provides evidence for an acid-base role, in addition to its long-accepted electrophilic role; A coenzyme with dual catalytic machinery. J Am Chem Soc 125:12732-12738, 2003.
Arjunan P, N Nemeria, A Brunskill, K Chandrasekhar, M Sax, Y Yan, F Jordan, JRGuest and W Furey. Structure of the pyruvate dehydrogenase multienzyme complex E1 component from Escherichia coli at 1.85 Å resolution., Biochemistry 41:5213-5221, 2002.

William Furey, PhD

Journal Articles

Koharudin L, W Furey and A Gronenborn.  Novel fold and carbohydrate specificity of the potent anti-HIV cyanobacterial lectin from oscillatoria agardhii.  J Biol Chem 286:1588-1597, 2011.
Nemeria N, P Arjunan, K Chandrasekhar, M Mossad, K Tittmann, W Furey and F Jordan.  Communication beween thiamin cofactors in the Escherichia coli pyruvate dehydrogenase complex E1 component active center:  Evidence for a "direct pathway" between the 4'-aminopyridine N1' atoms.  J Biol Chem 285:11197-11209, 2010.
Matei E, A Zheng, W Furey, J Rose, C Aiken and A Gronenborn.  Anti-HIV activity of defective cyanovirin-N mutants is restored by dimerization.  J Biol Chem 285:13057-13065, 2010.
Jordan F, P Arjunan, S Kale, N Nemeria and W Furey.  Multiple roles of mobile active center loops in the E1 component of the Escherichia coli pyruvate dehydrogenase complex:  Linkage of protein dynamics to catalysis.  J Mol Cat B:  Enzymatic 61:14-22, 2009.
Kale S, G Ulas, J Song, G Brudvig, W Furey and F Jordan. Efficient coupling of catalysis and dynamics in the E1 component of escherichia coli pyruvate dehydrogenase multienzyme complex. Proc Natl Acad Sci 105(4):1158-1163, 2008.
Arjunan P, M Sax, A Brunskill, K Chandrasekhar, N Nemeria, S Zhang, F Jordan and W Furey. A thiamin-bound, pre-decarboxylation reaction intermediate analogue in the pyruvate dehydrogenase E1 subunit induces large-scale disorder-to-order transformations in the enzyme and reveals novel structural features in the covalently bound adduct. J Biol Chem 281:15296-15303, 2006.
Jordan F, N Nemeria, S Zhang, Y Yan, P Arjunan and W Furey. Dual catalytic apparatus of the thiamin diphosphate coenzyme: Acid-base via the 1’.4’ iminopyrimidine tautomer along with its electrophilic role. J Am Chem Soc 125:12732-12738, 2003.
Nemeria N, P Arjunan, A Brunskill, F Sheibani, W Wei, Y Yan, S Zhang, F Jordan and W Furey. Histidine 407, a phantom residue in the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex, activates reductive acetylation of lipoamide on the E2 subunit. An explanation for conservation of active sites between the E1 subunit and transketolase. Biochemistry 41(52):15459-15467, 2002.
Arjunan P, N Nemeria, A Brunskill, K Chandrasekhar, M Sax, Y Yan, F Jordan, J Guest and W Furey. Structure of the pyruvate dehydrogenase multienzyme complex E1 component from Escherichia coli at 1.85Å resolution. Biochemistry 41(16):5213-5221, 2002.
Furey W and S Swaminathan. PHASES-95: A Program Package for Processing and Analysing Diffraction Data From Macromolecules in Methods in Enzymology: Macromolecular Crystallography, Part B, Vol 277, eds. C. Carter & R. Sweet, Academic Press, Orlando, Fl., pp 590-620, 1997.

Matthew J. Whitley, PhD

Journal Articles

Whitley MJ, Arjunan P, Nemeria NS, Korotchkina LG, Park YH, Patel MS, Jordan F and Furey W.  Pyruvate dehydrogenase complex deficiency is linked to regulatory loop disorder in the alphaV138M variant of human pyruvate dehydrogenase. J Biol Chem. 293: 13204-13213, 2018.
Boatz JC, Whitley MJ, Li M, Gronenborn AM and van der Wel PCA. Cataract-associated P23T γD-crystallin retains a native-like fold in amorphous-looking aggregates formed at physiological pH. Nat Commun. 8:15137, 2017.
Whitley MJ, Xi Z, Bartko JC, Jensen MR, Blackledge M and Gronenborn AM. A combined NMR and SAXS analysis of the partially folded cataract-associated V75D γD-Crystallin. Biophys J. 12:1135-114, 2017.
Xi Z, Whitley MJ and Gronenborn AM. Human βB2-Crystallin forms a face-en-face dimer in solution: An integrated NMR and SAXS study. Structure 25:496-505, 2017.
 
McDonald LR, Whitley MJ, Boyer JA and Lee AL. Colocalization of fast and slow timescale dynamics in the allosteric signaling protein CheY. J Mol Biol. 425:2372-2381, 2013.
Whitley MJ, Furey W, Kollipara S and Gronenborn AM. Burkholderia oklahomensis agglutinin is a canonical two-domain OAA-family lectin: structures, carbohydrate  binding and anti-HIV activity. FEBS J. 280:2056-2067, 2013.
Whitley MJ and Lee AL. Exploring the role of structure and dynamics in the function of chymotrypsin inhibitor 2. Proteins 79:916-924, 2011.
 
Whitley MJ and Lee AL. Frameworks for understanding long-range intra-protein communication. Curr Protein Pept Sci. 10:116-127, 2009.