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Conformational changes in rhodopsin Example Lecture

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Biochemistry 38, 7938-7944. ... Biochemistry 38, 7945-7949; Langen ... and Engineered Cysteines in Cytoplasmic Loop 1. Biochemistry 40, 12472-12478. ... – PowerPoint PPT presentation

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Title: Conformational changes in rhodopsin Example Lecture


1
Conformational changes in rhodopsinExample
Lecture
  • Judith Klein-SeetharamanCo-Course Director
  • jks33_at_pitt.edu

2
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

3
Tips on Preparation of Your Lecture
4
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

5
Which slide is better?
6
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.

7
Rhodopsin
Member of the G protein coupled receptor family
Cytoplasmic
11-cis Retinal
Transmembrane
Disulfide Bond
Extracellular
Glycosylation
8
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.

9
Use visual aids as much as possible!
10
Function of Rhodopsin
Signal Transduction
hn
G-Protein (Sensitization)
Rhodopsin Kinase (Desensization)
Conformational Changes are at the Heart of
Rhodopsins Function.
11
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
12
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
13
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
14
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
15
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
16
Tertiary Structure Probes
EPR
EPR provides information on mobility and tertiary
interactions
17
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 ().
18
Proximity
EPR Spin-Spin Interactions
19
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?
20
Summary
Current Picture of Conformational Changes upon
Light Activation
IV
II
III
V
I
VI
VII
21
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.

22
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