1. TOWARDS THE EXPERIMENTAL VALIDATION OF A NEW MEMBRANE PROTEIN FOLDING MODEL : A report on my work in Dr.Judith Klein- Seetharaman’s lab from 1 st September 2005 to 31s t December 2005 Varsha Shridhar

2. The Two-stage Hypothesis for the Folding of Membrane Proteins Step 1- The transmembrane segments of membrane proteins form helices independently in the lipid environment Step 2- Individual helices make helix-helix contacts and come together to assemble the whole protein. Protein folding studies on bacteriorhodopsin strongly support this model. Bacteriorhodopsin helix fragments can associate in vitro to form the whole protein.

4. Studies on rhodopsin, however, do not support this model. Instead, they seem to indicate that tertiary contacts between residues in the loops connecting the helices and those in the helices themselves are important in the assembly of rhodopsin.

5. Thus, it is thought that, in the case of rhodopsin at least, long range interactions between loop and transmembrane residues are more important than the short range interactions between transmembrane helices. It is even possible that these long range interactions take place first, and somehow cooperatively induce the correct folding of the protein. The likely ‘folding core’ of rhodopsin has been predicted by various simulation methods- FIRST and Gaussian Network Modelling.

6. Approaches Planned to Prove the Long-Range Interactions Model: A) Investigate the stability of rhodopsin folding core mutants; B) Label cysteines inside and outside of the predicted folding core and attach biophysical probes to them, such as 19 F labels for NMR structural studies of the folding core

7. What I did during the past 4 months 1) A preliminary rhodopsin (Rho) stability assay 2) Purification of Rhodopsin and Labeling with different reagents (19F, TET and PDS) 3) Hydroxylamine assay

8. Why I did what I did 1) The Rho stability assay: Measures of Rho stability: a) The number of exposed cysteines on rhodopsin b) The protein’s resistance to denaturing agents. Thus, a) Find out the least [SDS] required to denature w/t Rho. Compare it with that required to denature mutant Rho. This could give an early idea about the relative stabilities of the two. b) We could also do the same by finding out the number of Cys labeled on a Rho sample treated with a given concentration of SDS.

9. Results of this experiment- 0.065% SDS is the least concentration reqd. to completely denature w/t Rho Rhodopsin is denatured by SDS. This is spectroscopically seen with the shifting of the 500nm peak of wildtype rhodopsin to 440nm

12. Kinetics of Denaturation can be studied by Cysteine Labeling Cysteine labeling in wildtype rhodopsin. By ~60 minutes after addition of PDS, both the outer cysteines are labeled in wildtype rhodopsin. Cycles refer to 5 minute intervals

13. 0.1% SDS is added at cycle 30.

14. Cysteine labeling in rhodopsin treated with 0.1% SDS. Each cycle refers to a time span of 5 minutes

15. 2) Purification of Rho from bovine retinae: 1 mg 200ug 600ug 200ug Unlabeled NEM labeled Unlabeled BV: 250uL 500uL 250uL 1.5 mL eppendorf BioRad Poly 5mL falcons Prep 2mL Affinity Chromatography column

17. 19F-labeled Rho using TET

18. The Hydroxylamine Story: Previous studies had shown that cysteines that are inaccessible in the dark state of rhodopsin become accessible upon light activation and leaving of the retinal. We would like to make use of this fact and label these cysteines with biophysical probes

19. Rhodopsin is not very stable after light-activation. Hence, we want to remove the retinal faster than through natural decay (to prevent aggregation of Rho) Hydroxylamine reacts with the retinal-Lys296 Schiff base releasing retinaloxime, which readily leaves the binding pocket It is crucial, however, to remove hydroxylamine efficiently during the rhodopsin preparation protocol, as it tends to react with the cysteine labels.

20. I spent the remainder of my rotation on establishing an assay to detect hydroxylamine. I obtained preliminary evidence suggesting that the reaction between hydroxylamine, cystine and PDS can be used as an assay.

21. Assays tried: A) With PDS:

22. With dTT:

23. With Cysteine: Absorbance spectra of sample containing 150uM PDS, 70mM hydroxylamine and 0.5uM Cysteine. Cycles refer to spectra taken at 10-minute intervals.

25. With Fe3+ and Sodium Salicylate: Fe 3+ reacts with salicylate to form a colored complex Fe 3+ (aq) + C 7 H 5 O 3 - (aq) Fe(C 7 H 5 O 3 ) 2+ (aq) Hydroxylamine would reduce Fe3+ to Fe2+, making this latter species unreactive with salicylate. Adding an excess of Fe3+ to a sample containing an unknown concentration of hydroxylamine would convert some of the Fe3+ to Fe2+. The remaining Fe3+ could then react with salicylate. The concentration of the product formed could then be checked by absorption spectroscopy, and the amount of hydroxylamine back-calculated

26. The graphs above show that the reaction between Ferric Nitrate and Sodium Salicylate is perfectly stoichiometric. The absorbance maximas (at 323 nm) lie in a straight line which passes through the origin.

27. As time increases, more Fe3+ is reduced to Fe2+ than is predicted by the chemical equation. This leads to lower and lower absorption maximas. Low sensitivity of test. Na.Sal refers to sodium salicylate. The concentration of Na.Sal in the first three samples is 100uM, and 1mM in the last.

28. Using NAD: Hydroxylamine was expected to reduce NAD to give NADH, both of which have distinctive spectra

30. Cystine: We then studied if hydroxylamine could perhaps reduce the disulfide bond in cystine, releasing two cysteines, which could then react with the PDS. 10uM Cystine is reacted with 0, 1, 5 and 10uM hydroxylamine. 50uM Cystine is reacted with 50uM of hydroxylamine. [PDS]= 150uM

31. And, even better…. The reaction between 50uM Cystine and 50uM hydroxylamine does not change with time.

32. A Final Recap: 1) The major part of my work consisted of attempting to find a sensitive assay for hydroxylamine. The cystine assay seems now to be the most promising. 2) Purification of rhodopsin protein was also carried out. Unlabelled rhodopsin, rhodopsin labeled with NEM (to block the two free cysteines on the extracellular domain of the protein) and rhodopsin labeled with 19F were prepared according to standard protocols, for use in future experiments.

33. 3) A smaller experiment carried out with SDS attempted to show the results of mutation on the stability of the protein, specifically on its denaturation kinetics. This study is incomplete. For the moment, it just shows that the minimum amount of SDS required to completely denature wildtype rhodopsin is 0.065%. This is important in the future NMR based experiments where addition of the right amount of denaturant will be important to denature the protein without contributing too much background noise.

34. Acknowledgements: http://www.cs.cmu.edu/~naveena/baby Judith Naveena David Harpreet Fernanda Kalyan Hussein Madhavi