Review of Questions on Conformational Changes in Membrane Receptor Rhodopsin
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Review of Questions on Conformational Changes in Membrane Receptor Rhodopsin and Overview of Bulk Biophysical Methods . Judith Klein-Seetharaman Co-Course Director [email protected] From Last Lecture: Tips on Lecture Preparation. Give a roadmap

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Review of Questions on Conformational Changes in Membrane Receptor RhodopsinandOverview of Bulk Biophysical Methods

Judith Klein-SeetharamanCo-Course Director

[email protected]


From last lecture tips on lecture preparation
From Last Lecture: Tips on Lecture Preparation Receptor Rhodopsin

  • 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)

Molecular Biophysics 3: Lecture 4


From last lecture summary
From last lecture: Summary Receptor Rhodopsin

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

Molecular Biophysics 3: Lecture 4


From last Lecture: Summary Receptor Rhodopsin

Current Picture of Conformational Changes upon Light Activation

Picture from proximity studies

IV

II

III

Picture from single cysteine studies

V

I

Rigid Body Movement

(Biophys. Evidence) vs.

Changes in Mobility (Crystallography)

VI

VII

Picture from crystal structure

Molecular Biophysics 3: Lecture 4


How would you study the significance of the conformational changes for function
How would you study the significance of the conformational changes for function?

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)

Molecular Biophysics 3: Lecture 4


From Structure/Dynamics to Function changes for function?

Effect of restricting conformational changes on function

Phosphor-

ylation

G

T

Activation

by RK

No Effect

No Effect

Abolished

Abolished

Enhanced

Abolished

Conformational changes need not be cooperative,

but can be segmental

Molecular Biophysics 3: Lecture 4


What other models of activation can you imagine
What other models of activation can you imagine? changes for function?

On blackboard

Molecular Biophysics 3: Lecture 4


Models for signal transduction mechanisms
Models for Signal Transduction Mechanisms changes for function?

Conformational changes

“frozen” dynamic signal

transduction model

mechanical signal transduction models

Kim S.-H., Prot.Sci., 3 (1994), pp. 159-165

Ottemann K.M., Science, 285 (1999), pp. 1751-1754

Molecular Biophysics 3: Lecture 4


Membrane Receptor Families changes for function?

GPCR

Chemo-/Phototaxis

EGFR

Integrins

Two types of receptors: Type I and Type II

Molecular Biophysics 3: Lecture 4


Objectives of this lecture
Objectives of this Lecture changes for function?

  • 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

Molecular Biophysics 3: Lecture 4


Overview of methods
Overview of methods changes for function?

On blackboard

Molecular Biophysics 3: Lecture 4


Organized by purpose
Organized by purpose changes for function?

  • To study biomolecular interactions

  • To study dynamics

  • To identify secondary structures

  • To identify oligomerization state

  • To identify tertiary structures

Molecular Biophysics 3: Lecture 4


Overview of bulk methods to study biomolecular interactions
Overview of bulk methods to study biomolecular interactions changes for function?

  • 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

Molecular Biophysics 3: Lecture 4


Overview of bulk methods to study dynamics
Overview of bulk methods to study dynamics changes for function?

  • 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)

Molecular Biophysics 3: Lecture 4


Techniques to identify secondary structure
Techniques to identify secondary structure changes for function?

  • CD spectroscopy

  • FTIR

  • X-ray crystallography

  • NMR spectroscopy

  • EPR spectroscopy (spin label)

Molecular Biophysics 3: Lecture 4


Techniques to identify oligomerization state
Techniques to identify oligomerization state changes for function?

  • Gel filtration

  • Static light scattering

  • Dynamic light scattering

  • SANS/SAXS

Elution volume [ml]

Molecular Biophysics 3: Lecture 4


Techniques to identify tertiary structure
Techniques to identify tertiary structure changes for function?

  • 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

Molecular Biophysics 3: Lecture 4


Comparison of qualitative tertiary structure descriptors complementarity of methods
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

    • Cysteine reactivity

  • Identification of specific tertiary interaction sites

    • EPR spin spin interaction

    • NMR (solution or solid-state) spin spin interaction

    • FRET

    • Disulfide or chemical crosslinking

Molecular Biophysics 3: Lecture 4


Limitations
Limitations complementarity of methods

On blackboard

Molecular Biophysics 3: Lecture 4


Limitations1
Limitations complementarity of methods

  • Stability

  • Resolution

  • Need for labeling

  • Information content

  • Native environment

  • Applicability

  • Quantities and source of biomolecules

Gather a list of limitations on the blackboard

Molecular Biophysics 3: Lecture 4


Stability
Stability complementarity of methods

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.

Molecular Biophysics 3: Lecture 4


2 resolution
2. Resolution complementarity of methods

In NMR: resolve signals from individual atoms

folded

unfolded

Unfolded proteins have a smaller chemical shift dispersion than folded proteins.

Molecular Biophysics 3: Lecture 4


2 resolution1
2. Resolution complementarity of methods

In crystallography: resolve atom positions

  • Example rhodopsin:

In rhodopsin: conformational changes may be smaller than resolution of the crystal

Molecular Biophysics 3: Lecture 4


2 resolution2
2. Resolution complementarity of methods

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.

Molecular Biophysics 3: Lecture 4


2 resolution3
2. Resolution complementarity of methods

Time resolution

Time scales of biological processes vary from fs to days.

Molecular Biophysics 3: Lecture 4


3 need for labeling
3. Need for labeling complementarity of methods

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

Molecular Biophysics 3: Lecture 4


4 information content
4. Information content complementarity of methods

  • Semi-quantitative methods

    • Limited information only

  • X-ray crystallography

    • Snap shots only

  • Cryo-EM

    • Lower resolution

Molecular Biophysics 3: Lecture 4


5 native environment
5. Native environment complementarity of methods

  • In vitro studies

    • Lacks molecular crowding of the cell

  • Membrane proteins

    • Lipid bilayer

  • X-ray crystallography

    • Not in solution

Molecular Biophysics 3: Lecture 4


6 applicability
6. Applicability complementarity of methods

  • 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)

Molecular Biophysics 3: Lecture 4


Applicability x ray
Applicability: X-ray complementarity of methods

Crystallography of the dark-light transition in rhodopsin

Crystals dark

Crystals light

Applicability is also tied in with resolution

Molecular Biophysics 3: Lecture 4


Applicability x ray1
Applicability: X-ray complementarity of methods

So why are we now getting crystals?

2I36

2I37

Molecular Biophysics 3: Lecture 4


Applicability x ray2
Applicability: X-ray complementarity of methods

So why are we now getting crystals?

Alternative explanation: crystal packing artifacts stabilize conformations that are not stable in solution.

Molecular Biophysics 3: Lecture 4


Applicability x ray3
Applicability: X-ray complementarity of methods

Crystallography of the dark-light transition in rhodopsin

Crystals dark

Crystals light

Applicability is also tied in with resolution

Molecular Biophysics 3: Lecture 4


So let s just do nmr with rhodopsin
So, let’s just do NMR with rhodopsin! complementarity of methods

Molecular Biophysics 3: Lecture 4


When would you do NMR with a protein? complementarity of methods

Molecular Biophysics 3: Lecture 4


Nmr spectroscopy
NMR spectroscopy complementarity of methods

  • Size

  • Stability

  • Sample homogeneity

  • Need for labeling

  • Quantities and source of biomolecules

General limitations

Molecular Biophysics 3: Lecture 4


Example Solution NMR of DAGK complementarity of methods

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

Molecular Biophysics 3: Lecture 4


Example Solution NMR of Rhodopsin complementarity of methods

It’s a headache.

Molecular Biophysics 3: Lecture 4


What is signal 1? complementarity of methods

How can you test your hypothesis?

Molecular Biophysics 3: Lecture 4


Assignment of Signal 1 complementarity of methods

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:

Molecular Biophysics 3: Lecture 4


Traditional Solution NMR Approaches complementarity of methods

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, 3409-13.

Molecular Biophysics 3: Lecture 4


Your questions
Your questions complementarity of methods

  • If you find time, pls. go through the document that has all the questions and write an answer

Conformational Changes in Rhodopsin

Molecular Biophysics 3: Lecture 4


Summary of this lecture
Summary of this Lecture complementarity of methods

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

Molecular Biophysics 3: Lecture 4


A request
A request complementarity of methods

Please send word files to Oznur if at all possible, especially with the questions.

Molecular Biophysics 3: Lecture 4


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