Conformational changes in rhodopsin Example Lecture

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Conformational changes in rhodopsinExample
Lecture

• Judith Klein-SeetharamanCo-Course Director
• jks33_at_pitt.edu

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Objectives of this Lecture

• Give you tips on preparation of your lecture
• Introduction to visual system
• Light-induced conformational changes in rhodopsin
• Dark-state dynamics in rhodopsin
• Open questions

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Tips on Preparation of Your Lecture
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Objectives of this Lecture
Always give a roadmap!

• Give you tips on preparation of your lecture
• Introduction to visual system
• Light-induced conformational changes in rhodopsin
• Dark-state dynamics in rhodopsin
• Open questions

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Which slide is better?
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Rhodopsin

• Rhodopsin is a G protein coupled receptor
• 7 transmembrane helices
• binds 11-cis retinal
• two glycosylation sites
• Disulfide bond very important for folding
• The extracellular and transmembrane domains are
  structurally tightly coupled.

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Rhodopsin
Member of the G protein coupled receptor family
Cytoplasmic
11-cis Retinal
Transmembrane
Disulfide Bond
Extracellular
Glycosylation
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Rhodopsin

• Rhodopsin is a G protein coupled receptor
• 7 transmembrane helices
• binds 11-cis retinal
• two glycosylation sites
• Disulfide bond very important for folding
• The extracellular and transmembrane domains are
  structurally tightly coupled.

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Use visual aids as much as possible!
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Function of Rhodopsin
Signal Transduction
hn
G-Protein (Sensitization)
Rhodopsin Kinase (Desensization)
Conformational Changes are at the Heart of
Rhodopsins Function.
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Function of Rhodopsin
Title
Signal Transduction
Subtitle
Basic Architecture of a Slide
hn
Image
G-Protein (Sensitization)
Text
Rhodopsin Kinase (Desensization)
Conformational Changes are at the Heart of
Rhodopsins Function.
Conclusion Line
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General Approach
Study of Conformational Changes in Rhodopsin

• Single Cysteine Mutants
• Tertiary Structure Probes
• Double Cysteine Mutants
• Proximity Relationships

Cysteine Mutagenesis Provides Unique Attachment
Site for Biophysical Probes
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General Approach
Study of Conformational Changes in Rhodopsin
Its okay to have text if you need it

• Single Cysteine Mutants
• Tertiary Structure Probes
• Double Cysteine Mutants
• Proximity Relationships

Cysteine Mutagenesis Provides Unique Attachment
Site for Biophysical Probes
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Biophysical Probes
Study of Conformational Changes in Rhodopsin
Rho
SH
Identify Secondary Structure Elements Relative
Orientations of Helices Aqueous/Membrane
Boundary Qualitative Indicators for Tertiary
Structure Conformational Changes
S
S
Rho
EPR Spectroscopy
N
.
O
Absorbance Spectroscopy
N
Rho
S
S
Different probes provide different types of
information
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Tertiary Structure Probes
Reactivity of single cysteine mutants
4,4-
Dithiodipyridine
(a)
Dark, R-SH
Thiopyridone
Rho
Rho

N
S
S
SH

Thiopyridone
Rho
SR
S
(b)
Light, R-SH
Tertiary structure and light-induced changes
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Tertiary Structure Probes
EPR
EPR provides information on mobility and tertiary
interactions
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Accessibility with EPR vs. cysteine reactivity
Mobility and accessibility of the R1 side chain
in the sequence 59-75. The mobility of the R1
side chain measured by the inverse of the central
resonance line width, H-1 (). The accessibility
to collision with molecular oxygen () and with
NiEDDA (). The concentration of NiEDDA was 20 mM,
and for O2 was that in equilibrium with air. The
dotted line has a period of 3.6 residues. The
function e for the surface (exposed) and mobile
residues ().
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Proximity
EPR Spin-Spin Interactions
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Proximity
Rates of Disulfide Bond Formation in Double
Cysteine Mutants
S
S
SH
HS
pH Increase
Rho
Rho
What would you conclude from this result?
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Summary
Current Picture of Conformational Changes upon
Light Activation
IV
II
III
V
I
VI
VII
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References

• Main Klein-Seetharaman, J. (2002) Dynamics in
  Rhodopsin. ChemBioChem 3, 981-986.
• Slides 15, 17 Klein-Seetharaman, J., Hwa, J.,
  Cai, K., Altenbach, C., Hubbell, W.L. and
  Khorana, H.G. (1999) Single Cysteine Substitution
  Mutants at Amino Acid Positions 55-75, the
  Sequence Connecting the Cytoplasmic Ends of Helix
  I and II in Rhodopsin Reactivity of the
  Sulfhydryl Groups and their Derivatives
  Identifies a Tertiary Structure that Changes Upon
  Light-Activation. Biochemistry 38, 7938-7944.
• Slide 16, 17 Altenbach, C., Klein-Seetharaman,
  J., Hwa, J., Khorana, H.G. and Hubbell, W.L.
  (1999) Structural Features and Light-Dependent
  Changes in the Sequence 59-75 Connecting Helices
  I and II in Rhodopsin A Site-Directed Spin
  Labeling Study. Biochemistry 38, 7945-7949
  Langen
• Slide 18 Farrens, D.L., C. Altenbach, K. Yang,
  W.L. Hubbell, H.G. Khorana, Requirement of
  rigid-body motion of transmembrane helices for
  light activation of rhodopsin. Science, 1996.
  274(5288) p. 768-70.
• Slide 19 Klein-Seetharaman, J., Hwa, J., Cai,
  K., Altenbach, C., Hubbell, W.L. and Khorana,
  H.G. (2001) Probing the Dark State Tertiary
  Structure in the Cytoplasmic Domain of Rhodopsin
  Proximities Between Amino Acids Deduced from
  Spontaneous Disulfide Bond Formation between
  Cys316 and Engineered Cysteines in Cytoplasmic
  Loop 1. Biochemistry 40, 12472-12478.

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