Dr. Betty Jean Gaffney —FSU Biological Science Faculty Member -->
BIOLOGICAL SCIENCE
FACULTY MEMBER

Dr. Betty Jean Gaffney

Office: C315 MAG Lab
Office: (850) 644-8547
Lab: MAG Lab
Fax: (850) 645-8447
Mail code: 4295
E-mail: gaffney@bio.fsu.edu


Professor, Florida State University, 1996-present
B.S. 1961 (Chemistry), Stanford
Ph.D. 1966 (Chemistry), Stanford
NIH Postdoctoral Fellowship 1966, Tohoku University, Sendai Japan
Varian Assoc. Postdoctoral Fellowship 1967
Research Associate, Stanford 1968-1973
NIH Research Career Development Award, 1975-80
Assistant Professor to Professor, Johns Hopkins University, 1974-1996.
Fellow of the Biophysical Society, 2009.
Graduate Faculty Status

Curriculum Vitae
List of Publications

Research and Professional Interests:

A. Overview

I have been fascinated with “reading the biological lipid code” throughout my career. The code extends from physical properties of lipid arrays all the way to enzymes that use membrane components as substrates- both of which render cell membranes dynamic. The goal is to find the messages in these dynamics. I contributed to chemical synthesis of nitroxide spin labels at the beginning of my career and applied EPR (electron paramagnetic resonance) spectroscopy to reveal the lifetime of lipid conformations in synthetic lipid bilayers, during a post-doc period in H.M. McConnell’s lab. Early in my faculty career at Johns Hopkins, I pursued similar studies culminating in discovery (and naming) of the “sub-transition” that occurs between low temperature phases of saturated lipid bilayers (1) and in synthesis of spin-labeled, bifunctional crosslinking reagents (2). Membrane components are constantly remodeled to signal the state of a cell, and to maintain fluidity. In the early 1980s (pre-cloning), the lipid remodeling lipoxygenases (LOX) were available in abundance only from soybeans and animal reticulocytes. While I proposed to examine, by EPR, how the non-heme iron center in LOX responds to lipid substrate binding, without a structure of the active site, this was a black box. I improved LOX purification and initiated a project with crystallographers J. Boyington and M. Amzel. By 1993, we published (Science) the inaugural structure of the 100 kDa soybean lipoxygenase. Happily, it has become the prototype of the lipoxygenase fold (PF00305). Like other non-heme iron enzymes, EPR spectra of ferric LOX have several forms (3). In order to account quantitatively for overlapping line shapes, in depth reevaluation of simulating spin 5/2 EPR spectra was made, with quantum chemist, Harris Silverstone (4). These themes can all be found in my present studies that examine the ways lipoxygenases interact with unsaturated lipids.

1. S. C. Chen, J. M. Sturtevant and B. J. Gaffney (1980) "Scanning Calorimetric Evidence for a Third Phase Transition in Phosphatidylcholine Bilayers", Proc. Natl. Acad. Sci. USA, 77: 5060-5063. PMC349995

2. B. J. Gaffney, G. L. Willingham and R. S. Schepp (1983) "Synthesis and Membrane Interactions of Spin Label Bifunctional Reagents", Biochemistry, 22: 881-892. PMID: 6301528.

3. B.J. Gaffney (1996) "Lipoxygenases: Structural Principles and Spectroscopy" Annu. Revs. Biophys. Biomol. Struct., 25:.431-59. PMID: 8800477.

4. B. J. Gaffney and H. J. Silverstone (1993) "Simulation of the EMR Spectra of High Spin Iron in Proteins", in Biological Magnetic Resonance, Vol. 13: EMR of Paramagnetic Molecules, L.J. Berliner and J. Reuben, Eds, Plenum N.Y., pp 1-57. PMC2860145.

B.            Contributions in more detail

1. Structure and Mechanism of Lipoxygenase

Two types of lipoxygenase were known early on: 5-LOX and 15-LOX, the numbers referring to the carbon bearing –OOH in products from arachidonic acid. Pathways to mediators of inflammation differ for the two products. We solved the 15-LOX structure, others showed 5-LOX had the same overall protein fold and iron center. Finding determinates of specificity guides our recent studies. By advanced pulsed EPR (ACERT resource, Cornell), we determined the location and dynamics of a lipid, spin labeled on the head group, in complex with soybean LOX (a). Trilateration between five site-directed spin labels, and to the substrate

                           Fig6Asimple.png

Fig 1 Cyan ellipse: distribution of head group locations outside blue helices. Iron center: rust, red (water).

analog, revealed the head group on the surface of the protein, near one proposed portal to the active site (Figure 1). EPR in my lab also showed the head group moving fairly freely, consistent with a spread of distance determinations. We earlier had computationally docked linoleic acid into 15- LOX (b, d) with the polar end emerging from the protein as found in (a). Evidence that a turn of pi-helix in a LOX surface helix contributes flexibility when substrate binds resulted from further spin labeling (c). Constructs, from which over 40 spin labeled 15-LOXs (soybean) were derived, are deposited with Addgene (https://www.addgene.org/Betty_Gaffney/).

a.  B.J. Gaffney, M.D. Bradshaw, S. Frausto, F. Wu, J.H. Freed and P. Borbat. (2012) “Locating a lipid at the portal to the lipoxygenase active site” Biophys. J., 103: 2134-2144. PMC3512035.

b.  G. Coffa, A.N. Imber, B.C. Maguire, G. Laxmikanthan, C. Schneider, B.J. Gaffney, and A.R. Brash (2005) "On the Relationships of Substrate Orientation, Hydrogen Abstraction and Product Stereochemistry in Single and Double Dioxygenations by Soybean Lipoxygenase-1 and Its Ala542Gly Mutant", J. Biol. Chem. 280: 38756-38766. PMC1351262.

c. M.D. Bradshaw and B.J. Gaffney (2014) “Fluctuations of an exposed pi-helix involved in lipoxygenase substrate recognition” Biochemistry, 53: 5102-5110. PMC4131896.

d. B.J. Gaffney (2014)  “Connecting Lipoxygenase Function to Structure by EPR” Accounts of Chemical Research, 47: 3588-3595. PMC 4270396.

2. Continued Support of Crystallography Projects on Lipoxygenase (LOX)

A flurry of papers with JC Boyington and LM Amzel followed our first LOX structure paper in Science (a). Later, I contributed dockings of substrate to a crystallography study by Howard Grimes and Chul-Hee Kang of 4 isoforms in soybean (b). It has been difficult to obtain LOX structures with a polyunsaturated lipid bound, because oxygen is a co-substrate. But a group in Barcelona crystallized the exported LOX of P. aeruginosa, and found a phospholipid acyl chain (non-substrate) in the active site. I looked at EPR spectra of the iron (ferric) center in this bacterial lipoxygenase. The spectrum is almost identical with one of the two spectra exhibited by soybean LOX (c). This identifies the ligand geometry of more rhombic symmetry as that associated with substrate binding. How the ROOH gets to the iron center, when another lipid is already bound, remains a puzzle.

a. J.C. Boyington, B. J. Gaffney, and L.M. Amzel (1993) "The Three-Dimensional Structure of an Arachidonic Acid 15-Lipoxygenase", Science, 260: 1482-1486. PMID 8502991.

b. B. Youn, G.E. Sellhorn, R.J. Mirchel, B.J. Gaffney, H.D. Grimes, C.H. Kang (2006) "Crystal Structures of Vegetative Soybean Lipoxygenase VLX-B and VLX-D, and comparisons with seed isoforms, LOX-1 and LOX-3” PROTEINS: Structure, Function, and Bioinformatics 65: 1008-1020. PMID 17022084.

c. A. Garreta, S. Val-Moraes, Q. García-Fernández, M. Busquets, C. Juan, A. Oliver, A. Ortiz, B.J. Gaffney, I. Fita, À. Manresa and X. Carpena (2013) “Structure and Interaction with Phospholipids of a Prokaryotic Lipoxygenase from Pseudomonas aeruginosa”, FASEB J, 27: 4811–4821. PMID: 23985801.

3.  Metalloprotein EPR Spectroscopy at Higher Magnetic Fields

At Florida State, I have compared the EPR spectra from a variety of metalloproteins at frequencies 9.4 and 94 GHz. The higher frequency is particularly suitable for manganese and copper (a-c). From EPR, the metal center in manganese lipoxygenase is predicted to be similar to that in the iron enzymes (a). Also, 94 GHz spectra of catalase and iron and copper transferrins were measured and analyzed by simulations (b, c).

a. B.J. Gaffney, C. Su and E.H. Oliw (2001) "Assignment of EPR Transitions in a Manganese-Containing Lipoxygenase and Prediction of Local Structure" Appl. Magn. Res., 21: 411-422. PMID 16518455.

b. B.J. Gaffney, B.C. Maguire, R.T. Weber and G.G. Maresch (1999) "Disorder at Metal Sites in Proteins: a High Frequency EMR Study" Appl. Magn. Res., 16: 207-222.

c. B.J. Gaffney (2009) "High Resolution EPR of Mononuclear Iron Proteins", Chapter 6 in Biological Magnetic Resonance, Vol 28.  High Resolution EPR: Applications to Metalloenzymes and Metals in Medicine, G. Hanson and L.J. Berliner eds., Springer Pub., pp. 233-268. PMID 20428459.

4.  Quantitative Assignment of EPR Transitions in Iron Proteins

My lab has examined EPR of several non-heme iron proteins, including phenylalanine hydroxylase (a, b), transferrins (b, c), and lipoxygenase (d). Quantitative evaluation of the low-temperature EPR spectra of these is intriguing and complex because the energy level separations are similar to the microwave energy applied, in X-band EPR. The complexity of manganese lipoxygenase EPR spectra at X-band is illustrated in this figure (from ref 1d above). 
                                              Fig7LeftSide.png

Each dot in (A) represents intensity from one molecular orientation in the magnetic field. These intensities sum to give the components in observed spectra (B). (My colleague, Harris Silverstone invented the style of diagrams like (A).) Multi-component EPR spectra are observed for each of the proteins studied, raising questions about the fraction of functional iron centers. Simulation, not integration, is required to provide answers. Our simulation model (b, c) takes a distribution in zero-field energies as the origin of unusual EPR line shapes of these proteins. Questions about the fraction of functional iron centers is particularly important for LOX because the enzyme cycles ferric/ferrous during catalysis (d), and also, iron centers in expressed LOX may not be fully loaded. Recently, these simulations have been important in showing that only a single equivalent of lipid hydroperoxide is required to convert all the iron in resting ferrous P. aeruginosa LOX to the ferric form (2.c. above).

a. L.M. Bloom, S.J. Benkovic and B.J. Gaffney (1986) "Characterization of Phenylalanine Hydroxylase", Biochemistry 25: 4204-4210. PMID 3019383.

b. A.S. Yang and B.J. Gaffney (1987) "Determination of Relative Spin Concentration in Some High-Spin Ferric Proteins Using E/D-Distribution in Electron Paramagnetic Resonance Simulations" Biophys. J., 51: 55-67. PMID 3026504.

c. B.J. Gaffney and H.J. Silverstone (1998) "Simulation Methods for Looping Transitions" J. Magn. Res., 134: 57-66. PMID 9740731.

d. B.J. Gaffney, D.V. Mavrophilipos and K.S. Doctor (1993) "Access of Ligands to the Ferric Center in Lipoxygenase-1" Biophys. J., 64: 773-783. PMID 8386016.

5.  The Properties of Lipids in Biological Membranes

In early studies of lipid membrane structure (1970s), it was unclear if real biological membranes and lipid model bilayers had similar properties. I showed that the abnormal appearance of transformed fibroblasts does NOT indicate a global change in lipid properties from their normal counterparts, as determined with spin labeled fatty acids (a). On the other hand, mobility of phospholipid spin labels in the close-packed membranes of Sindbis virus changes dramatically when the E1/E2 proteins are removed by proteolysis (b). The existence of patches of membrane with specialized physical properties was foreshadowed by these landmark papers.

a. B.J. Gaffney (1975) "Fatty Acid Chain Flexibility in the Membranes of Normal and Transformed Fibroblasts", Proceedings of the National Academy of Sciences, 72: 664-668. PMID 164663.

b. B.M. Sefton and B. J. Gaffney (1974) "Effect of the Viral Proteins on Fluidity of the Membrane Lipids in Sindbis Virus", Journal of Molecular Biology, 90: 343-358. PMID4375723.

 A List of my publications can be found at:

http://www.ncbi.nlm.nih.gov/sites/myncbi/betty.gaffney.1/bibliography/40434843/public/?sort=date&direction=ascending

Selected Publications:

Other:
Bowers, C. R., V. Storhaug, C. E. Webster, J. Bharatam, A. Cottone III, R. Gianna, K. Betsey, and B.J. Gaffney (1999). Exploring protein surfaces and cavities in lipoxygenase and other proteins by hyperpolarized xenon-129 NMR. Journal of the American Chemical Society 121: 9370-9377. PMID: 16429610. Full text, PDF

Wu, F., L. J. Katsir, M. Seavy, B. J. Gaffney. 2003. Role of radical formation at Y193 in the allene oxide synthase domain of a lipoxygenase-AOS fusion protein from coral. Biochemistry 42: 6871-6880. PMID: 12779342.

Agarwalla, S., R. M. Stroud, and B. J. Gaffney. 2004. Redox reactions in the iron-sulfur cluster in a ribosomal RNA methyltransferase, RumA: optical and EPR studies. Journal of Biological Chemistry 279:34123-34129. PMID: 15181002.

Wu, F. Y., and B. J. Gaffney. 2006. Dynamic behavior of fatty acid spin labels within a binding site of soybean Lipoxygenase-1. Biochemistry 45:12510-12518. PMID: 17029406.

Gaffney, B. J. 2009. EPR of mononuclear iron proteins. Pages 233-268 in Biological Magnetic Resonance, Vol 28. High Resolution EPR: Applications to Metalloenzymes and Metals in Medicine, G. Hanson and L. J. Berliner, eds. Springer Verlag. ISBN 978-0-64655-6.  See Figure 9 in color.

Gaffney, B. J., M. D. Bradshaw, S. Frausto, F. Wu, J. H. Freed, and P. Borbat. 2012. Locating a lipid at the portal to the lipoxygenase active site. Biophysical Journal 103:2134-2144. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3512035.
See also Supplemental information.

Garreta, Val-Moraes, Garcia-Fernandez, Busquets, Juan, Oliver, Ortiz, Gaffney, Fita, Manresa and Carpena. 2013. Structure and interaction with phospholipids of a prokaryotic lipoxygenase from Pseudomonas aeruginosa FASEB J. 37: 4811-4821.

Bradshaw, M.D. and B.J. Gaffney. 2014. “Fluctuations of an exposed pi-helix involved in lipoxygenase substrate recognition” Biochemistry  53: 5102-5110. DOI: 10.1021/bi500768c.

Gaffney, B.J. 2014.  “Connecting Lipoxygenase Function to Structure by Electron Paramagnetic Resonance” Accounts of Chemical Research 47: 3588-3595. http://dx.doi.org/10.1021/ar500290r.

Graduate Students:

Bradshaw, Miles

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