1. Judith Klein-Seetharaman Co-Course Director jks33@pitt.edu
Review of Questions on Conformational Changes in Membrane Receptor Rhodopsin and Overview of Bulk Biophysical Methods
Judith Klein-Seetharaman Co-Course Director

2. From Last Lecture: Tips on Lecture Preparation
Give a roadmap
Give an overview of the field introducing your topic
Use visual aids as much as possible - but is okay to have text if needed
Basic architecture of a slide:
Title
Subtitle
Text and/or image
Conclusion line
Give appropriate references (papers, websites) for resources you used (especially images)
Have a summary at the end of the lecture
Use my last lecture as a template for font sizes and colors (pls. do not use your own style)
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Molecular Biophysics 3: Lecture 4

3. From last lecture: Summary
Conformational changes in rhodopsin
Introduction to rhodopsin
Seven transmembrane helical bundle with posttranslational modifications and ligand retinal bound
General approach: cysteine mutagenesis and attachment of biophysical probes
Biophysical methods applied to single cysteines
Mobility with EPR spectroscopy
Secondary structure from EPR mobility
Helix orientation from EPR mobility
Tertiary structure from EPR hyperfine lines
Tertiary structure from cysteine reactivity rates with absorbance spectroscopy
Aqueous / membrane boundary from EPR collision frequency (O2 versus NiEDDA)
Exposed loops from cysteine reactivity
Biophysical methods applied to double cysteines
Proximity from disulfide bond formation rates
Proximity from EPR spin spin interactions
Conformational changes by comparing the above parameters for different states – here dark versus light
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Molecular Biophysics 3: Lecture 4

4. Changes in Mobility (Crystallography)
From last Lecture: Summary
Current Picture of Conformational Changes upon Light Activation
Picture from proximity studies IV II
Picture from single cysteine studies
III
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.). V I
Rigid Body Movement
(Biophys. Evidence) vs.
Changes in Mobility (Crystallography) VI
VII
Picture from crystal structure
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Molecular Biophysics 3: Lecture 4

5. Molecular Biophysics 3: Lecture 4
How would you study the significance of the conformational changes for function?
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Molecular Biophysics 3: Lecture 4

6. Molecular Biophysics 3: Lecture 4
How would you study the significance of the conformational changes for function? Prevent conformational changes and see if the protein still functions correctly (here binds proteins of the signal transduction cascade)
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Molecular Biophysics 3: Lecture 4

7. Conformational changes need not be cooperative,
From Structure/Dynamics to Function
Effect of restricting conformational changes on function
Phosphor-
ylation G T
Activation
by RK
No Effect
No Effect
Abolished
Abolished
Finally, what’s the functional role of these conformational changes anyways??? Effects of restricting them: … Read bottom line.
Enhanced
Abolished
Conformational changes need not be cooperative,
but can be segmental
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Molecular Biophysics 3: Lecture 4

8. What other models of activation can you imagine?
On blackboard
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Molecular Biophysics 3: Lecture 4

9. Models for Signal Transduction Mechanisms
Conformational changes
“frozen” dynamic signal
transduction model
mechanical signal transduction models
Kim S.-H., Prot.Sci., 3 (1994), pp
Ottemann K.M., Science, 285 (1999), pp
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Molecular Biophysics 3: Lecture 4

10. Two types of receptors: Type I and Type II
Membrane Receptor Families
GPCR
Chemo-/Phototaxis
EGFR
Integrins
Why do we need methods to determine distance constraints in full-length receptors?
Two types of receptors: Type I and Type II
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Molecular Biophysics 3: Lecture 4

11. Objectives of this Lecture
Overview of bulk methods and what they are used for
Use the topic conformational changes in membrane receptors to gather a list of biophysical methods (your homework)
Decide on who will write summaries on what methods
Limitations of biophysical bulk methods
Go through your questions on conformational changes in rhodopsin – will only have time for one
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Molecular Biophysics 3: Lecture 4

12. Molecular Biophysics 3: Lecture 4
Overview of methods
On blackboard
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Molecular Biophysics 3: Lecture 4

13. Molecular Biophysics 3: Lecture 4
Organized by purpose
To study biomolecular interactions
To study dynamics
To identify secondary structures
To identify oligomerization state
To identify tertiary structures
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Molecular Biophysics 3: Lecture 4

14. Overview of bulk methods to study biomolecular interactions
Identification (mass spectrometry, HPLC, absorbance, fluorescence, radioactivity)
Kinetics (absorbance, fluorescence, Biacore)
Size (gel filtration, light-scattering…)
Identification of specific interaction sites (fluorescence quench, spin spin interactions…)
Accessibility
NMR spectroscopy
X-ray crystallography
Cryo-EM
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Molecular Biophysics 3: Lecture 4

15. Overview of bulk methods to study dynamics
Global methods:
Methods to identify secondary structure (changes)
Methods to identify tertiary structure (changes)
Methods to quantify oligomerization state
Semi-quantitative methods:
Accessibility at specific sites
Motion at specific sites
Identification of specific tertiary contacts
Atomic resolution methods:
Accessibility by H/D exchange + NMR or mass spec
NMR spectroscopy (structure and dynamics)
X-ray crystallography (mostly structure)
Cryo-electron microscopy (mostly structure)
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Molecular Biophysics 3: Lecture 4

16. Techniques to identify secondary structure
CD spectroscopy
FTIR
X-ray crystallography
NMR spectroscopy
EPR spectroscopy (spin label)
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Molecular Biophysics 3: Lecture 4

17. Techniques to identify oligomerization state
Gel filtration
Static light scattering
Dynamic light scattering
SANS/SAXS
Elution volume [ml]
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Molecular Biophysics 3: Lecture 4

18. Techniques to identify tertiary structure
Intrinsic Fluorescence
Fluorescence (fluorescence label)
EPR spectroscopy
FTIR
Raman
CD spectroscopy
(far-UV – secondary structure, near-UV – immobilization of aromatic residues in tertiary structure)
Dye binding (e.g. ANS)
NMR spectroscopy (1H,13C,15N,2H,19F)
Solution
Solid state
X-ray
Cryo-EM
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Molecular Biophysics 3: Lecture 4

19. Molecular Biophysics 3: Lecture 4
Comparison of qualitative tertiary structure descriptors – complementarity of methods
Motion and tertiary structure indicators
EPR single labels
Cysteine reactivity
Fluorescence labels
NMR labels
Accessibility
EPR collision
19F NMR
1H/2H titrations
Identification of specific tertiary interaction sites
EPR spin spin interaction
NMR (solution or solid-state) spin spin interaction
FRET
Disulfide or chemical crosslinking
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Molecular Biophysics 3: Lecture 4

20. Molecular Biophysics 3: Lecture 4
Limitations
On blackboard
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Molecular Biophysics 3: Lecture 4

21. Limitations Stability Resolution Need for labeling Information content
Gather a list of limitations on the blackboard
Stability
Resolution
Need for labeling
Information content
Native environment
Applicability
Quantities and source of biomolecules
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Molecular Biophysics 3: Lecture 4

22. Molecular Biophysics 3: Lecture 4
Stability
Crystallography of the dark-light transition in rhodopsin
Crystals dark
Crystals light
The conformational changes upon illumination destroy the crystals and crystals grown in the light do not diffract.
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Molecular Biophysics 3: Lecture 4

23. Molecular Biophysics 3: Lecture 4
2. Resolution
In NMR: resolve signals from individual atoms
folded
unfolded
Unfolded proteins have a smaller chemical shift dispersion than folded proteins.
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Molecular Biophysics 3: Lecture 4

24. Molecular Biophysics 3: Lecture 4
2. Resolution
In crystallography: resolve atom positions
Example rhodopsin:
In rhodopsin: conformational changes may be smaller than resolution of the crystal
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Molecular Biophysics 3: Lecture 4

25. Certain questions require very high resolution.
In crystallography: resolve atom positions
Example rhodopsin: conformational changes smaller than resolution of the crystal structure
Example low barrier hydrogen bonds:
Certain questions require very high resolution.
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Molecular Biophysics 3: Lecture 4

26. Time scales of biological processes vary from fs to days.
2. Resolution
Time resolution
Time scales of biological processes vary from fs to days.
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27. Molecular Biophysics 3: Lecture 4
3. Need for labeling
Signal versus background
Intrinsic Fluorescence
no tryptophans, no fluorescence
NMR
No 15N, 13C, no selective excitation
FTIR, Raman
Large background signals
Resonance Raman
Needs a chromophore
Attachment of probes (EPR, fluorescence, etc.)
Perturbation of the native environment by large size
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28. Molecular Biophysics 3: Lecture 4
4. Information content
Semi-quantitative methods
Limited information only
X-ray crystallography
Snap shots only
Cryo-EM
Lower resolution
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Molecular Biophysics 3: Lecture 4

29. Molecular Biophysics 3: Lecture 4
5. Native environment
In vitro studies
Lacks molecular crowding of the cell
Membrane proteins
Lipid bilayer
X-ray crystallography
Not in solution
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30. Molecular Biophysics 3: Lecture 4
6. Applicability
X-ray
Crystallization conditions empirical
NMR
Limited by size
Limited by labeling ability
Transient intermediates
adequate time-resolution
Sensitivity to conformational changes
small changes need to be detectable (size of label often much larger than distance change)
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Molecular Biophysics 3: Lecture 4

31. Applicability is also tied in with resolution
Applicability: X-ray
Crystallography of the dark-light transition in rhodopsin
Crystals dark
Crystals light
Applicability is also tied in with resolution
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Molecular Biophysics 3: Lecture 4

32. Molecular Biophysics 3: Lecture 4
Applicability: X-ray
So why are we now getting crystals?
2I36
2I37
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Molecular Biophysics 3: Lecture 4

33. Molecular Biophysics 3: Lecture 4
Applicability: X-ray
So why are we now getting crystals?
Alternative explanation: crystal packing artifacts stabilize conformations that are not stable in solution.
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Molecular Biophysics 3: Lecture 4

34. Applicability is also tied in with resolution
Applicability: X-ray
Crystallography of the dark-light transition in rhodopsin
Crystals dark
Crystals light
Applicability is also tied in with resolution
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Molecular Biophysics 3: Lecture 4

35. So, let’s just do NMR with rhodopsin!
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Molecular Biophysics 3: Lecture 4

36. When would you do NMR with a protein?
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Molecular Biophysics 3: Lecture 4

37. Molecular Biophysics 3: Lecture 4
NMR spectroscopy
General limitations
Size
Stability
Sample homogeneity
Need for labeling
Quantities and source of biomolecules
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Molecular Biophysics 3: Lecture 4

38. Example Solution NMR of DAGK
1H,15N-HSQC spectrum of a 120 aa long membrane protein in DPC micelles
Diacylglycerol kinase:
Charles R. Sanders, Frank Sonnichsen (2006) Solution NMR of membrane proteins: practice
and challenges. Magn. Reson. Chem. 2006; 44: S24–S40
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Molecular Biophysics 3: Lecture 4

39. Example Solution NMR of Rhodopsin
It’s a headache.
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40. What is signal 1? How can you test your hypothesis? 11/25/2018
Molecular Biophysics 3: Lecture 4

41. Molecular Biophysics 3: Lecture 4
Assignment of Signal 1
NMR Spectroscopy
Black: original spectrum, red: C-terminus, green: N-terminus (after AspN cleavage)
An enzyme was used to cleave off the C-terminus at the site indicated below:
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Molecular Biophysics 3: Lecture 4

42. Traditional Solution NMR Approaches
Problems with full-length membrane proteins in detergents
Size – is not the only problem (Trosy does not work for helical membrane proteins)
Conformational exchange – fluctuations in the detergent micelle environment lead to fast relaxation thus signal decay
Spin diffusion – cannot deuterate samples from mammalian cells
Problem: Traditional assignment strategies using triple resonance experiments (13C,15N,1H) don’t work
Klein-Seetharaman et al. (2004) PNAS 101,
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Molecular Biophysics 3: Lecture 4

43. Your questions
Conformational Changes in Rhodopsin
If you find time, pls. go through the document that has all the questions and write an answer
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Molecular Biophysics 3: Lecture 4

44. Summary of this Lecture
Complementarity of biophysical bulk methods
Example Membrane Receptor Conformational Changes
Stability
Resolution
Need for labeling
Information content
Native environment
Applicability
Quantities and source of biomolecules
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Molecular Biophysics 3: Lecture 4

45. Molecular Biophysics 3: Lecture 4
A request
Please send word files to Oznur if at all possible, especially with the questions.
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Molecular Biophysics 3: Lecture 4