NSLS Activity Report 2006 - Brookhaven National Laboratory

More documents

Recommendations

Info

BEAMLINE X6A PUBLICATION M. Nadella, M.A. Bianchet, S.B. Gabelli, J. Barrila, and L.M. Amzel, "Structure and <strong>Activity</strong> of the Axon Guidance Protein MICAL," Proc. Natl. Acad. Sci. USA,102(46),16830-5 (2005). FUNDING <strong>National</strong> Institutes of Health MORE INFORMATION L.M. Amzel Department of Biophysics and Biophysical Chemistry Johns Hopkins University School of Medicine mario@neruda.med.jhmi.edu Neurons are required to make path-finding decisions throughout their development and are guided to their final targets by a variety of environmental cues. Semaphorins are a family of guidance molecules that act as repellents in a variety of axon development processes. Repulsive guidance by Semaphorins is mediated through their interaction with Plexins, a family of transmembrane receptors. Biochemical and genetic analysis indicates that a large multi-domain cytosolic protein, MICAL (Molecule Interacting with Cas- L), is required for the repulsive axon guidance mediated by the interaction of Semaphorins and Plexins. MICAL proteins contain a large aminoterminal FAD-binding domain (MICAL fd ), followed by a series of protein-protein binding domains. MICAL fd is of great interest since it offers a novel link between redox reactions and an axon guidance response. Using x-ray crystallography, we determined the structure of murine MICAL1 FADbinding domain, MICAL fd (Figure 1). Authors (from left) L.M. Amzel, S.B. Gabelli, M.A. Bianchet, and M. Nadella A REDOX REACTION IN AXON GUIDANCE: STRUCTURE AND ENZYMATIC ACTIVITY OF MICAL M. Nadella, M.A. Bianchet, S.B. Gabelli, and L.M. Amzel Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine During development, neurons are guided to their final targets by external cues. MICAL, a large multidomain cytosolic protein, is a downstream signaling molecule required for repulsive axon guidance. We have determined the structure of the N-terminal FAD-binding domain of MICAL to 2.0 Å resolution. This structure shows that MICAL fd is structurally similar to aromatic hydroxylases and amine oxidases. We obtained biochemical data that show that MICAL fd is a flavoenzyme that, in the presence of NADPH, reduces molecular oxygen to H 2 O 2 . We propose that the H 2 O 2 produced by this reaction may be one of the signaling molecules involved in axon guidance by MICAL. 2-76 MICAL fd is a mixed α/β globular protein that contains a Rossmann β-α-β fold, two conserved FADbinding motifs, and a third conserved sequence motif. The first conserved GXGXXG dinucleotide binding motif resides within the Rossman fold. The second conserved GD motif has been observed in flavoprotein hydroxylases and forms part of a strand and a helix. A search of known structures reveals that the MICAL fd protein is most similar to aromatic hydroxylases, especially the p-hydroxybenzoate hydroxylase (pHBH) from P. fluorescens (rms 1.79 Å for 199 out of 484 aligned α-Carbons). The strong structural similarity of MICAL fd to PHBH suggests that the two proteins might have similar enzymatic activities. Since purified MICAL fd contains oxidized FAD, reduction of the cofactor was tested using either NADH (nicotinamide adenine dinucleotide) or NADPH (nicotinamide adenine dinucleotide phosphate). Although no net reduction of the FAD was detected, a steady, time-dependent oxidation of reduced nicotinamide dinucleotide was observed (Figure 2). This observation suggested that enzyme-bound FADH 2 was formed but was then reoxidized by oxygen. The resulting production of H 2 O 2 was confirmed by monitoring its formation with horseradish peroxidase in a coupled spectrophotometric assay (Figure 2). The rate of the reaction is over 10 times faster with 200 µM NADPH than with 200 µM NADH, suggesting that MICAL is probably an NADPH-dependent enzyme. The observation of this enzymatic activity can be explained by one of three cases. First, H 2 O 2 is the physiological product of the enzyme and is a component of the avoidance signal. Second, as with other FAD hydroxylases, in the absence of
substrate the hydroperoxide form of the enzyme (the MICAL fd -FADH-O 2 H intermediate) decomposes, producing hydrogen peroxide and oxidized enzyme-bound FAD (Scheme 1). Third, the enzyme may actually be an amine oxidase in which the FADH 2 is reoxidized by molecular oxygen, and the reduction by NADPH is a fortuitous, non-specific reaction. Although this last case is unlikely, discrimination among these pos- Figure 1. Ribbon representation of the tertiary structure of MICAL fd . MICAL fd is a mixed α/β globular protein composed of two sub-domains of different sizes linked by two β-strands. Subdomain-1 is colored in magenta and subdomain-2 is colored in light blue. The observed FAD molecule is colored in yellow. The large sub-domain (subdomain-1; residues 1 to 226 and 373 to 484) contains the two known FAD sequence motifs (residues 84 to 114 and 386 to 416) and a third conserved motif typically found in hydroxylases (residues 212 to 225). The fi rst motif is part of a Rossmann β-α-β fold (β1-α5-β2 in MICAL fd ). This is a sequence commonly found in FAD and NAD(P)Hdependent oxidoreductases. The second motif, which contains a conserved GD sequence in hydroxylases, forms part of a strand and a helix. In MICAL fd this second conserved sequence makes contacts with the ribose moiety of FAD. 2-77 sibilities requires further experimentation. The synthesis of a specific metabolite and the production of reactive oxygen species have been previously proposed as possible mechanisms of MICAL signaling. We have shown here that the FAD-binding domain of MICAL can generate at least the second kind of signals: MICAL reduces molecular oxygen using NADPH to produce H 2 O 2 . Figure 2. Kinetics of NADPH oxidation and H 2 O 2 production. The absorbance peak at 340 nm is characteristic of reduced NAPDH and the peak at 560 nm the concentration of H 2 O 2 . The experiment ran for fi ve minutes after the addition of the enzyme. The inset shows the initial rates as a function of NADPH concentration. Scheme 1 LIFE SCIENCE
Page 1 and 2:
NATIONAL SYNCHROTRON LIGHT SOURCE A
Page 3 and 4:
NATIONAL SYNCHROTRON LIGHT SOURCE 2
Page 5 and 6:
NSLS Summer Sunday Draws 650 Visito
Page 7 and 8:
CHAIRMAN'S INTRODUCTION Chi-Chang K
Page 9 and 10:
USERS' EXECUTIVE COMMITTEE REPORT C
Page 11 and 12:
SCIENCE AT THE NSLS Kendra Snyder N
Page 13 and 14:
Spin-Lattice Interaction in the Qua
Page 15 and 16:
SOFT CONDENSED MATTER AND BIOPHYSIC
Page 17 and 18:
grows. The magnesium also is attrac
Page 19 and 20:
Chicago, Liangtao Li and Jerry Kapl
Page 21 and 22:
STRUCTURE OF AN E. COLI MEMBRANE PR
Page 23 and 24:
for 15 minutes at 180 °C, a 1 x 2
Page 25 and 26:
Said collaborator Eliot Rosen, a ra
Page 27 and 28:
AT THE NSLS, SCIENTISTS WORKING TOW
Page 29 and 30:
NANOPARTICLE ASSEMBLY ENTERS THE FA
Page 31 and 32:
Crystal structure of VCBP3 V1V2 sol
Page 33 and 34: National Laboratory; and Mati Meron
Page 35 and 36: X-RAY ANALYSIS METHOD CRACKS THE 'L
Page 37 and 38: the enzyme and the cofactor binds a
Page 39 and 40: ticles (Figure 1). The nearest-neig
Page 41 and 42: is about four times larger. The ver
Page 43 and 44: Figure 1. Ball model of the UO 2 (1
Page 45 and 46: of barium sulfate species formed up
Page 47 and 48: observed in the normal-state of the
Page 49 and 50: and calculate the contribution to t
Page 51 and 52: energy gap, responsible for suppres
Page 53 and 54: 20%. Correspondingly, the Fe octahe
Page 55 and 56: We fit the data in Figure 1 by modi
Page 57 and 58: of the 1812 RF buckets of the ring
Page 59 and 60: from the laser, we allow enough tim
Page 61 and 62: scopic and microscopic characteriza
Page 63 and 64: suboxic boundary and a concomitant
Page 65 and 66: intensity of the CaP shoulder (Figu
Page 67 and 68: effectively combined to elucidate A
Page 69 and 70: ecomes evident through comparison o
Page 71 and 72: forming an electrostatic interactio
Page 73 and 74: promises the possibility of being a
Page 75 and 76: etween TPP and related compounds is
Page 77 and 78: (Figure 2). The negative charge on
Page 79 and 80: spots were observed in control brai
Page 81 and 82: malaria parasite to the red blood c
Page 83: of the resulting complex revealed t
Page 87 and 88: of AlkB-∆N11 matches that in othe
Page 89 and 90: Figure 3. In catalysis, this array
Page 91 and 92: that form a central hydrophobic pat
Page 93 and 94: structural differences suggest that
Page 95 and 96: occurs between subunits located tow
Page 97 and 98: We used x-ray absorption near edge
Page 99 and 100: tonically as ξ increases (Figure 1
Page 101 and 102: These results demonstrated that a m
Page 103 and 104: position of the x-ray beam, pulse-h
Page 105 and 106: geneity ranges are observed with no
Page 107 and 108: PEGylated fluoroalkyl side chains r
Page 109 and 110: interface by annealing bilayer film
Page 111 and 112: β-phase takes place, giving rise t
Page 113 and 114: persion was followed by static and
Page 115 and 116: 411TH BROOKHAVEN LECTURE ‘ SHININ
Page 117 and 118: space. Its delivery of this materia
Page 119 and 120: NSLS RESEARCHERS PRODUCE A PRAISEWO
Page 121 and 122: in Farmingville, New York. She inve
Page 123 and 124: egan with three days of lectures an
Page 125 and 126: Participants in the 2006 “Take ou
Page 127 and 128: While Dehmer was optimistic about t
Page 129 and 130: Next, a talk on the “Engineering
Page 131 and 132: Energy Secretary Samuel Bodman (fro
Page 133 and 134: with majors ranging from history to
Page 135 and 136:
described the first measurements of
Page 137 and 138:
Visitors gather around the liquid n
Page 139 and 140:
high-level degree in the physics fi
Page 141 and 142:
Spotlight Awards The Spotlight awar
Page 143 and 144:
A) B) Figure 1. X-ray fl uorescence
Page 145 and 146:
BNL WINS R&D 100 AWARD FOR X-RAY FO
Page 147 and 148:
2005 Hans-Jürgen Engell Prize The
Page 149 and 150:
2006 NSLS TOURS 1/4/2006 Karen Agos
Page 151 and 152:
2006 NSLS SEMINARS 1/11/2006 Interi
Page 153 and 154:
2006 NSLS SEMINARS 6/22/2006 Strate
Page 155 and 156:
2006 NSLS WORKSHOPS 4/4/2006 X6A Wo
Page 157 and 158:
NATIONAL SYNCHROTRON LIGHT SOURCE O
Page 159 and 160:
NSLS ADVISORY COMMITTEES GENERAL US
Page 161 and 162:
ACCELERATOR DIVISION REPORT James B
Page 163 and 164:
On-Axis Field (gauss) VTF Test Arti
Page 165 and 166:
The NSLS has an aggressive preventi
Page 167 and 168:
tific workshops, including “Synch
Page 169 and 170:
ently the last one available in the
Page 171 and 172:
functions, particularly at the nano
Page 173 and 174:
A new Proposal Oversight Panel (POP
Page 175 and 176:
SAFETY REPORT Andrew Ackerman ESH&Q
Page 177 and 178:
BUILDING ADMINISTRATION REPORT Bob
Page 179 and 180:
ing were cleaned, repaired, and pai
Page 181 and 182:
FACILITY FACTS & FIGURES The Nation
Page 183 and 184:
Beamline Source Technique Energy Ra
Page 185 and 186:
Page 187 and 188:
Page 189 and 190:
NSLS BOOSTER PARAMETERS NSLS LINAC
Page 191 and 192:
X-RAY STORAGE RING PARAMETERS AS OF
Page 193 and 194:
PUBLICATIONS
Page 195 and 196:
NSLS USERS Beamline U1A S Buzby, M
Page 197 and 198:
A Nambu, J Graciani, J Rodriguez, Q
Page 199 and 200:
Beamline X1A2 M Feser, B Hornberger
Page 201 and 202:
C Mandel, D Gebauer, H Zhang, L Ton
Page 203 and 204:
Beamline X7A E Anokhina, Y Go, Y Le
Page 205 and 206:
T Moldoveanu, Q Liu, J Tocilj, M Wa
Page 207 and 208:
Beamline X10A A Cisneros, G Mazzant
Page 209 and 210:
S Kamtekar, R Ho, M Cocco, W Li, S
Page 211 and 212:
Z Zhang, P Fenter, S Kelly, J Catal
Page 213 and 214:
E Selvi, Y Ma, R Aksoy, A Ertas, A
Page 215 and 216:
Y Suh, M Carroll, R Levy, G Bisogni
Page 217 and 218:
S Park, E DiMasi, Y Kim, W Han, P W
Page 219 and 220:
G Da, J Lenkart, K Zhao, R Shiekhat
Page 221 and 222:
J Kaste, B Bostick, A Friedland, A
Page 223 and 224:
X Chen, K Tenneti, C Li, Y Bai, R Z
Page 225 and 226:
G Meinke, P Bullock, A Bohm, Crysta
Page 227 and 228:
D Koller, G Ediss, L Mihaly, G Carr
show all

NSLS Activity Report 2006 - Brookhaven National Laboratory

Create successful ePaper yourself

Delete template?

Save as template?