Diapositive 1

1
PhD project Solution state NMR structure
determination of PorA and PorH, the major Porins
of Corynebacterium glutamicum
By Parthasarathi Rath

• Principal Investigator
• Prof A. Milon, NMR Spectroscopy, IPBS/CNRS,
  Toulouse, France
• In Collaboration With
• Dr F. Bernhard, CBMR, Inst. Biophys. Chem.,
  Frankfurt, Germany
• Dr M. Daffé, IPBS/CNRS, Toulouse, France

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Objectives

• Expression purification in Corynebacterium
  glutamicum
• 2. Expression purification in cell free
  expression system
• 3. Functional reconstitution in detergent
  micelles and in lipid bilayers
• (biophysical and biochemical studies,
  oligomerisation states
• and structure function relationship)
• 4. 3D structure resolution of PorA and PorH by
  solution state NMR
• 5. 3D structure in bilayers by solid state NMR

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Introduction

• Corynebacterium glutamicum
• member of the mycolic-acid containing
  actinomycetes
• aerobic and non-sporulating
• genes are orthologous to number of human
  pathogens, such as
• Corynebacterium diphtheriae, Mycobacterium
  tuberculosis and Mycobacterium leprae
• (can be targeted as anti-tubercular agent)
• industrial production of L-glutamate (
  appetite enhancer), L-lysine (animal food
  additive)
• at the rate of about 1 Megaton/year
• Challenges
• Amino acid efflux properties ?
• Understanding cell wall complexity ?

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Cell Envelope of Gram-Negative, Gram-Positive and
Corynebacterineae
Porins
Lipoteichoic and Teichoic acids
Corynebacterineae
Gram-negative
Gram-positive

• Mycolic acids
• long chain a-alkyl, ß-hydroxyl fatty acids
• play a crucial role in determining the
  fluidity
• and permeability of the cell wall

• Porins
• major channel-forming proteins
• ion-selective passage of small hydrophillic
  
• molecules (substrates / products /
  antibiotics)
• important for aminoacid excretion ??

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Type of Porins

• PorA
• 45aa (5KDa) long polypeptide
• cation-selective channel G 5,5nS in 1M KCl
• major hydrophilic pathway, transport of
  antibiotics (Amp, Kan, Stp )
• no N-terminal extension (Post-translationally
  modified ??)
• (Reference J. Bacteriology, 2003,185,
  p.4779-4786)
• 2) PorH
• 57aa long polypeptide (12KDa dimer)
• cation-selective channel G 2,5nS in 1M KCl
• co-transcribed with PorA
• no N-terminal extension (the export pathway ??)
• (Reference BBA, 2005, 1715, p. 25-36)

Only PorB structure is known PDB no 2VQG

• 3) PorB and PorC
• Anion-selective channels with N-terminal
  extension
• (Reference Mol. Microbiol. 2003, 50, p.
  1295-1308)

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First objective Expression and Purification of
PorA and PorH(Cloning and Sequencing)
Vector constructions
pK18 E.coli based plasmid pBL1 C.glutamicum
plasmid

• pXMJ19
• pTAC (promotor)
• laq Iq
• CmR (resistance to chloramphenicol)
• MCS (multiple cloning site)

PorA N-His
n-MASRGSHHHHHHHHIEGRENVYEFLGNLDVLSGSGLIGYVFDFLGASS
KWAGAVADLIGLLG -c
PorH C-His n-DLSLLKETLGNYETFGGNIGTALQSIPTLLDSILNFF
DNFGDLADTTGENLDNFSSIEGRASRGSHHHHHHHH -c
IEGR Factor Xa cleavage site
Expression
PorA / PorH Toxic for E. coli C.
glutamicum - PorA/-PorH

• Homologous Expression
• Post-translational modification (if any)

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Extraction and Purification
TritonX-100
CHCl3 CH3OH
Yield/L culture 12mg PorA 4mg PorH
Yield/ L culture 11.2mg PorA
7.6mg PorH
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Cell Free SynthesisDr. Frank Bernhard. CBMR,
Frankfurt

• Low expression level in E. coli
• Toxic effects
• Inefficient transport

Expression PorA / PorH Toxic for E. coli
Reference Proteomics 2008, 8, 3933-3946
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Prospectives

• Express, Label (13C / 15N) in Minimal Medium for
  NMR
• To characterize the purified protein in MALDI-TOF
• in-vitro synthesis of pET28a-PorA / -PorH
  constructs

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Thanks

• Prof. Alain Milon, IPBS/CNRS, Toulouse
• (NMR and protein - membrane interactions)
• Prof J. Czaplicki
• Dr P. Demange
• Dr V. Gervais
• Dr I. Muller
• Dr V. Reat
• Dr O. Saurel
• P. Ramos

Future Collaborator Dr F.
Bernhard, CBMR, Frankfurt

• Dr M. Daffé, IPBS/CNRS, Toulouse
• (Mycobacterial envelopes structure,
  biosynthesis and functions)
• Dr M. Tropis
• Emilie Huc