"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