70S Ribosome from thermus thermophilus

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70S Ribosome from thermus thermophilus
2.5 MegaDaltons
Selmer et al., Science, 313, 1935-1942 (2006)
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Why use NMR?
Dynamics
Example BPTI 30-51 folding intermediate
Interactions
Example F1 alpha - OSCP complex
Things that dont crystallize
Example RDS3
David Neuhaus MRC Laboratory of Molecular
Biology Cambridge, U.K.
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Why use NMR?
Dynamics
Example BPTI 30-51 folding intermediate
Interactions
van Mierlo, Darby, Keeler, Neuhaus Creighton J.
Mol. Biol. (1993), 229, 1125-1146.
Example F1 alpha - OSCP complex
Things that dont crystallize
Example RDS3
David Neuhaus MRC Laboratory of Molecular
Biology Cambridge, U.K.
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Bovine Pancreatic Trypsin Inhibitor
time
oxidation level
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Bovine Pancreatic Trypsin Inhibitor disuphide
folding pathway

(30-51)
R
others
(5-55)
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Bovine Pancreatic Trypsin Inhibitor disuphide
folding pathway

(30-51)
R
others
(5-55)
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Bovine Pancreatic Trypsin Inhibitor disuphide
folding pathway
Oxidative coupling of cysteines by reaction with
glutathione
Non-native 2-disulphide species are necessarily
involved in rearrangements between native
2-disulphide species
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Bovine Pancreatic Trypsin Inhibitor disuphide
folding pathway
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BPTI (30-51) intermediate 15N relaxation analysis
30-51
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Why use NMR?
Dynamics
Example BPTI 30-51 folding intermediate
Interactions
Example F1 alpha - OSCP complex
Things that dont crystallize
Carbajo, Kellas, Yang, Runswick, Montgomery,
Walker Neuhaus J. Mol. Biol. (2007), 368,
310-318.
Example RDS3
David Neuhaus MRC Laboratory of Molecular
Biology Cambridge, U.K.
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OSCP N-terminal domain
Hydrophobic groove thought to interact with
N-terminal tail(s) of F1 a subunits
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15N HSQC shift mapping addition of F1? 1-25 to
labelled OSCP-NT
?? HN gt average s.d.
?? HN gt average
HN signal disappears
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13C HSQC shift mapping addition of F1? 1-25 to
labelled OSCP-NT
?? HC gt average s.d.
?? HC gt average
Predicted groove
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OSCP-NT F1?(1-25) NOESY spectra
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OSCP-NT F1?(1-25) NOESY spectra
OSCP-NT F1? (1-25) Filtered 2D NOESY spectrum
OSCP-NT F1? (1-25) Filtered 3D NOESY spectrum
Something on F1? at 0.9ppm
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OSCP N-terminal domain
F1? 1-25
17
OSCP N-terminal domain - unlabelled
F1? 1-25 Leu 17 13C615N
Cross-peak remains
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OSCP N-terminal domain - 13C 15N labelled
Cross-peak must be due to NOE linking OSCP Ala
25 ? to Leu17 ? and Ile16 ?
F1? 1-25 Leu 17 13C615N
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OSCP-NT F1?(1-25) complex structure
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Why use NMR?
Dynamics
Example BPTI 30-51 folding intermediate
van Roon, Loening, Obayashi, Yang, Newman,
Hernandez, Nagai Neuhaus Proc. Nat. Acad. Sci.
U.S.A. (2008), 105, 9621-9626.
Interactions
Example F1 alpha - OSCP complex
Things that dont crystallize
Example RDS3
David Neuhaus MRC Laboratory of Molecular
Biology Cambridge, U.K.
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pre-mRNA Splicing
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U2 snRNP
interaction partners as yet unknown
wont crystallise
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RDS3 - ordered regions and zinc clusters
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NMR ensemble calculated with ARIA
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Its a knot
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Three sorts of calculation give the same knotted
structure
Two sorts of calculation give the same knotted
structure
ARIA
ATNOSCANDID
Manual assignment
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Building up the Triquetra motif
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26
61
58
30
73
33
49
76
11
86
46
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The central ?-triangle
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Holding it together
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Other points of interest

• Its active
• RDS3 expressed in E. coli, exactly as prepared
  for NMR, is active in a yeast splicing assay.

• Its very stable
• Thermal unfolding of RDS3 is incomplete even at
  90C, and is reversible.
• Treatment with EDTA causes irreversible and
  complete unfolding.

• Fusions dont affect folding
• RDS3 is initially expressed as an N-terminal
  fusion protein.
• The fusion protein binds three zinc ions, showing
  it is folded despite the fusion.
• Others have shown that a C-terminal fusion is
  active in splicing.

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Acknowledgements
BPTI 30-51 folding intermediate
Carlo van Mierlo, Nigel Darby, James Keeler, Tom
Creighton
F1 alpha - OSCP complex
Rodrigo Carbajo, Fiona Kellas, Ji-Chun Yang, Mike
Runswick, Martin Montgomery, John Walker
RDS3
Marike van Roon, Niko Loening, Ji-Chun Yang, Andy
Newman, Helena Henandez, Carol Robinson, Kiyoshi
Nagai
David Neuhaus MRC Laboratory of Molecular
Biology Cambridge, U.K.
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ATP synthase
Mitochondrial Matrix
ADP Pi
Peripheral stalk
F1
Central Stalk
H
H
H
e-
pH and voltage gradient
I
III
IV
Fo
Inner mitochondrial membrane
H
Intermembrane Space
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Building up the Triquetra motif
23
26
61
58
30
73
33
49
76
11
86
46
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ATPase peripheral stalk

• Acts as a stator to counter the rotation of F1

Oligomycin Sensitivity Conferral Protein

• Crystallisation has been difficult (perhaps due
  to 3-fold positional disorder)

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OSCP N-terminal domain - 13C 15N labelled
F1? 1-25 Ile 11 2H10
Cross-peak remains
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OSCP N-terminal domain - 13C 15N labelled
F1? 1-25 Leu 12 2H10
Cross-peak remains
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RDS3
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Its a knot from the back as well
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Far-UV CD measurements
0.2 mg/ml Rds3
10 mM Pi pH 7.2, 100 mM NaCl,1 mM DTT
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Defining the clusters
113Cd-1H Correlation
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NMR ensemble calculated with ARIA
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Defining the clusters
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Electrostatic surface
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Hydrophobic residues
Conclusion this protein is part of a complex
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Why use NMR?
Dynamics
Example BPTI 30-51 folding intermediate
Interactions
Example F1 alpha - OSCP complex
Things that dont crystallize
Example RDS3
Tour de Force
Sec A
David Neuhaus MRC Laboratory of Molecular
Biology Cambridge, U.K.
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ATPase subunit composition
F1 (catalytic) domain
a3, b3, g, d, e
peripheral stalk
OSCP, F6, (part of) b, d
Fo (membrane) domain
a, (part of) b, c10
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Protein Interactions by NMR
N-terminal domain of OSCP interacting with
N-terminal tails of ATPase enzyme
Rodrigo J. Carbajo Fiona A. Kellas Mike Runswick
John Walker David Neuhaus
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ATPase crystal structures
Abrahams, J.P., Leslie, A.G.W., Lutter, R.
Walker, J.E. Nature 370, 621-628 (1994)
Stock, D., Leslie, A.G.W. Walker, J.E. Science
286, 1700-1705 (1999)
Gibbons, C., Montgomery, M.G., Leslie, A.G.W.
Walker, J.E. Nat. Struct. Biol. 7, 1055-1061
(2000)
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ATP synthase enzyme
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OSCP N-terminal domain - 13C 15N labelled
F1? 1-25 Leu 17 13C615N
Cross-peak remains
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Other components of the Knotome
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