1. Conformational changes in rhodopsin Example Lecture
Judith Klein-Seetharaman Co-Course Director

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
11/30/2018
Molecular Biophysics 3: Lecture 2

3. Tips on Preparation of Your Lecture
11/30/2018
Molecular Biophysics 3: Lecture 2

4. Objectives of this Lecture
Always give a roadmap!
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
11/30/2018
Molecular Biophysics 3: Lecture 2

5. Molecular Biophysics 3: Lecture 2
Which slide is better?
11/30/2018
Molecular Biophysics 3: Lecture 2

6. Molecular Biophysics 3: Lecture 2
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.
11/30/2018
Molecular Biophysics 3: Lecture 2

7. Rhodopsin Member of the G protein coupled receptor family
Cytoplasmic
11-cis Retinal
Transmembrane
Disulfide Bond
Extracellular
Glycosylation
11/30/2018
Molecular Biophysics 3: Lecture 2

8. Molecular Biophysics 3: Lecture 2
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.
11/30/2018
Molecular Biophysics 3: Lecture 2

9. Use visual aids as much as possible!
11/30/2018
Molecular Biophysics 3: Lecture 2

10. Function of Rhodopsin Signal Transduction hn G-Protein (Sensitization)
Rhodopsin Kinase (Desensization)
Conformational Changes are at the Heart of Rhodopsin’s Function.
11/30/2018
Molecular Biophysics 3: Lecture 2

11. Basic Architecture of a Slide Subtitle
Function of Rhodopsin
Title
Signal Transduction
Basic Architecture of a Slide
Subtitle hn
Image
G-Protein (Sensitization)
Text
Rhodopsin Kinase (Desensization)
Conformational Changes are at the Heart of Rhodopsin’s Function.
Conclusion Line
11/30/2018
Molecular Biophysics 3: Lecture 2

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
11/30/2018
Molecular Biophysics 3: Lecture 2

13. It’s okay to have text if you need it
General Approach
Study of Conformational Changes in Rhodopsin
It’s 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
11/30/2018
Molecular Biophysics 3: Lecture 2

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
Rho S S
EPR Spectroscopy N . O
Absorbance Spectroscopy
Rho N S S
Different probes provide different types of information
11/30/2018
Molecular Biophysics 3: Lecture 2

15. Tertiary structure and light-induced changes
Tertiary Structure Probes
Reactivity of single cysteine mutants
4,4’-
Dithiodipyridine
(a)
Dark, R’-SH
Rho SH
Rho S S N +
Thiopyridone
Rho S
SR’ +
Thiopyridone
(b)
Light, R’-SH
This slide shows an example of the results obtained probing tertiary structure by studying the reactivity of single cysteine mutants in the CP loop connecting helices I and II. The reaction is outlined here… On the x-axis you see.. On the y-axis you see… As you can see, there are dramatic differences in the rate at which cysteines in different – here even neighboring positions – react with 4-PDS, indicating differences in the tertiary structure depending on location. More interestingly, you can look at differences in the release rates at these positions when one compares rhodopsin in the dark and in the light. Both faster, and slower reactions were observed upon light-activation.
Tertiary structure and light-induced changes
11/30/2018
Molecular Biophysics 3: Lecture 2

16. Molecular Biophysics 3: Lecture 2
Tertiary Structure Probes
EPR
EPR provides information on mobility and tertiary interactions
11/30/2018
Molecular Biophysics 3: Lecture 2

17. Accessibility with EPR vs. cysteine reactivity
Mobility and accessibility of the R1 side chain in the sequence 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 ().
11/30/2018
Molecular Biophysics 3: Lecture 2

18. Proximity EPR Spin-Spin Interactions 11/30/2018
Molecular Biophysics 3: Lecture 2

19. What would you conclude from this result?
Proximity
Rates of Disulfide Bond Formation in Double Cysteine Mutants HS SH S S
pH Increase
Rho
Rho
Helix I
Helix II
Helix VII
“Helix VIII”
Helix VI
C-terminus
V61
C316
H65
L68
To further probe the tertiary structure in the CP domain, we then studied proximity relationships using disulfide crosslinking of double cysteine mutants. These are mutants with two free cysteines in their CP domain, which can be purified in their free suflhydryl form. Upon increasing the pH, these cysteines form disulfide bonds with rates indicative of their proximity. The result is shown here for disulfides exploring proximity between the I-II loop and the end of helix VII (imagine the helical bundle, where helix one closes the bundle on helices I and II). On the x-axis, on the y-axis. You can see that there are clearly peaks in the rates of disufldie bond formation, at 61, 65 and 68. The publication of the crystal structure has allowed me to compare these results with proximity observed in the crystal. I replaced the original amino acids in the crystal with the respective cysteines, and measured the S-S distance. The reciprocal is shown superimposed onto the rates of disulfide bond formation here. Very good match. However, these distances indicate that clearly there has to be motion that allows the two cysteines to come together to form a disulfide bond, thus reporting on motion, not only on pure proximity.
What would you conclude from this result?
11/30/2018
Molecular Biophysics 3: Lecture 2

20. Summary
Current Picture of Conformational Changes upon Light Activation IV II
III V I
Taken together with EPR spectroscopy, the results I have described to you have allowed us to come up with a comprehensive map of the tertiary structure in the CP domain and their changes upon light-activation. Explain… While this accounts for the most detailed description to date, these approaches cannot be expected to be quantiative. Therefore, I have explored an approach which has the potential to yield atomic resolution description of the tertiary structure: high-resolution solution NMR spectroscopy (since at the time X-ray crystallography seemed hopeless – today we know that it is possible, but still the problem remains that multiple crystal structures need to be solved, dark light protein-bound etc.). VI
VII
11/30/2018
Molecular Biophysics 3: Lecture 2

21. Molecular Biophysics 3: Lecture 2
References
Main: Klein-Seetharaman, J. (2002) Dynamics in Rhodopsin. ChemBioChem 3,
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,
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 Connecting Helices I and II in Rhodopsin: A Site-Directed Spin Labeling Study. Biochemistry 38, ; 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, (5288): p
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,
11/30/2018
Molecular Biophysics 3: Lecture 2

22. Use this presentation as a template for your presentation!
11/30/2018
Molecular Biophysics 3: Lecture 2