Conformational changes in rhodopsin Example Lecture

1. Conformational changes in rhodopsinExample Lecture Judith Klein-SeetharamanCo-Course Director jks33@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 Molecular Biophysics 3: Lecture 2

3. Tips on Preparation of Your Lecture Molecular Biophysics 3: Lecture 2

4. 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 Molecular Biophysics 3: Lecture 2

5. Which slide is better? Molecular Biophysics 3: Lecture 2

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. Molecular Biophysics 3: Lecture 2

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

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. Molecular Biophysics 3: Lecture 2

9. Use visual aids as much as possible! 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. Molecular Biophysics 3: Lecture 2

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

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

15. Tertiary Structure Probes Reactivity of single cysteine mutants 4,4’- Dithiodipyridine (a) Dark, R’-SH N Rho Rho + Thiopyridone S S SH Rho + Thiopyridone S SR’ (b) Light, R’-SH Tertiary structure and light-induced changes Molecular Biophysics 3: Lecture 2

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

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 (). Molecular Biophysics 3: Lecture 2

18. Proximity EPR Spin-Spin Interactions Molecular Biophysics 3: Lecture 2

19. Helix VI C-terminus Helix VII C316 L68 H65 V61 Helix II “Helix VIII” Helix I Proximity Rates of Disulfide Bond Formation in Double Cysteine Mutants S S HS SH pH Increase Rho Rho What would you conclude from this result? Molecular Biophysics 3: Lecture 2

20. Summary Current Picture of Conformational Changes upon Light Activation IV II III V I VI VII Molecular Biophysics 3: Lecture 2

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. Biochemistry38, 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. Biochemistry38, 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. Biochemistry40, 12472-12478. Molecular Biophysics 3: Lecture 2

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