Post on 21-Aug-2020
"Structural Studies of Photosystem II: Electron Crystallography &
Characterization of a Novel Chromophore"
Tina M. DreadenGeorgia Institute of Technology
July 2009
Outline
I. Electron crystallographya. Known structuresb. 2-D crystallization
II. Chromophorea. Significanceb. Previous workc. Data
Photosynthesis
6CO2 + 6H2O + light C6H12O6 + 6O2
2.9 Å resolution structure in T. elongatus
• 20 subunits• 35 chlorophyll a• 12 carotenoids• 25 integral lipids• 3 platostoquinones• Mn4Ca active site
Guskov, A. et al. (2009) Nat. Struct. Mol. Biol. 16, 334-342
Eukaryotic plant PSII
• Different light harvesting system (LHC-II, Cab proteins)
• Different extrinsic subunits
• ~ 1000 kDa
Boekema, E. J. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 175-179Ferreira, K. et al. (2004) Science 303, 1831-1837
Plant PSII at 8 Å resolution
• D1, D2, CP47, cyt. b-559• ~160 kDa monomer• Inactive
Rhee, K.-H. et al. (1997) Nature 396, 283-286Ferreira, K. et al. (2004) Science 303, 1831-1837
Cyanobacteria (3 Å resolution)
Plant (8 Å resolution)
Overview of 3-D structure determination
Important factors for 2-D crystallization of membrane proteins
1. Protein (purity, yield)2. Detergent 3. Lipid to protein ratio (LPR)4. Dialysis buffer (salts)5. Dialysis time6. Temperature
Schmidt-Krey, I. (2007) Methods 41, 417-426
2-D crystals of partial PSII complex
Nakazato K. et al. (1996) J. Mol. Biol. 257, 225-232
Near future goals
• Optimize crystal size & order• Temperature• Dialysis time• Salt concentration
Outline
I. Electron crystallographya. Known structuresb. 2-D crystallization
II. Chromophorea. Significanceb. Previous workc. Optical & HPLC data
Basic protein biochemistry
Proteins are composed of 20 natural amino acids
Post-translational modifications (PTM)
• Important functions in proteins– catalysis, regulation and protein degradation
• PSII– Phosphorylations1
– Oxidative and reductive modifications2, 3, 4
– Amine/ phenylhydrazine-binding residues5, 6
1. Vener, A. V. et al. (2001) J. Biol. Chem 276, 6959-69662. Anderson, L. B. et al. (2004) J. Am. Chem. Soc. 126, 8399-84053. Anderson, L. B. et al. (2002) Proc, Natl. Acad. Sci. U. S. A. 99, 14676-146814. Rexroth, S. et al. (2007) J. Biol. Chem. 282, 27802-278095. Ouellette, A. J. A. et al. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2204-22096. Anderson, L. B. et al. (2000) J. Biol. Chem. 275, 4920-4927
TPQ binds amines & hydrazines
R-CH2-NH2 + H2O + O2 R-C-H + NH3 + H2O2
=O
O
O
OH
O
NH-CH2-R
OH
H2N-CH2-R
+
O
OOH
HNH2N
phenylhydrazine
O
OHHN
+HN
Janes, S. M. et al. (1990) Science 248, 981-987
TPQ titration with hydrazines
Steinebach, V. et al. (1995) Anal. Biochem. 230, 159-166
pNPH
PH
(expanded)
untreated
Increase access to active site
Ouellette, A. J. A. et al. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2204-2209
14C-labeling: binding occurs close to active site
Ouellette, A. J. A. et al. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2204-2209
Coomassie 14C-detection
O
O
OH
O
NH-14CH2-R
OH
H2N-14CH2-R
+
PSII preparation & derivatization
O
OOH
HNH2N
phenylhydrazine
O
OH HN
+HN
Peptide digestion & chromophore purification
Trypsin
HPLC
nm200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600
mA
U
0
50
100
150
200
250
300
Near future goals
• Awaiting mass spectrometry results• Model peptides• NMR• Raman