PPT X-Ray Crystallography and It's Applications - Bharathidasan University

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X-Ray Crystallography X-Ray Crystallography

and and

It’s Applications It’s Applications

Dr. P. MURUGAN,

M.Sc., M.Phil., Ph.D.,

Assistant Professor and Principal i/c Department of Biochemistry,

Bharathidasan University Model College,

Vedharanyam - 614 810.

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Introduction Introduction

 Present basic concepts of protein structure Present basic concepts of protein structure

 Discuss why x-ray crystallography is used to Discuss why x-ray crystallography is used to determine protein structure

determine protein structure

 Lead through x-ray diffraction experiments Lead through x-ray diffraction experiments

 And present how to utilize experimental And present how to utilize experimental information to design structural models of information to design structural models of

proteins

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Introduction to Protein Structure:

“ “ The Crystallographer’s Problem” The Crystallographer’s Problem”

 What is the crystallographer’s What is the crystallographer’s problem?: Structural

problem?: Structural Determination!

Determination!

 Structure ~ Function Structure ~ Function

 Amino acids are strung together Amino acids are strung together on a carbon chain backbone.

on a carbon chain backbone.

 As a result:As a result:

 Can be described by the dihedral Can be described by the dihedral angles, called

angles, called φφ, , ψψ, and , and ωω angles. angles.

 Ramachandran PlotRamachandran Plot

 Note: the crystallographer is not Note: the crystallographer is not in the business of determining in the business of determining

molecular composition, but molecular composition, but

determining structural orientation determining structural orientation

of a protein.

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Introduction to:

X-Ray Crystallography X-Ray Crystallography

 x-rays are used to probe the x-rays are used to probe the protein structure:

protein structure:

 Why are x-rays used? Why are x-rays used?

 λ ~ Åλ ~ Å

 Why are crystals used to do x-ray Why are crystals used to do x-ray diffraction?

diffraction?

 Crystals are used because it helps Crystals are used because it helps amplify the diffraction signal.

amplify the diffraction signal.

 How do the x-rays probe the How do the x-rays probe the crystal?

crystal?

 x-rays interact with the electrons x-rays interact with the electrons surrounding the molecule and surrounding the molecule and

“reflect”. The way they are

reflected will be prescribed by the reflected will be prescribed by the orientation of the electronic

orientation of the electronic distribution.

distribution.

 What is really being measured? What is really being measured?

 Electron Density!!!Electron Density!!!

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Performing X-Ray Crystallography Experiments Performing X-Ray Crystallography Experiments

aka aka

“Just Do It”

 Bragg’s Law: Bragg’s Law:

 n n λ λ =2d =2d sin( sin( θ θ ) )

 Bragg's Law Applet

 X-Ray Diffraction X-Ray Diffraction apparatus.

apparatus.

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Performing X-Ray Diffraction Performing X-Ray Diffraction

 Resultant diffraction Resultant diffraction pattern from

pattern from

experimental setup experimental setup

 Diffraction pattern is Diffraction pattern is actually a Fourier

actually a Fourier Transform of the Transform of the

electron distribution electron distribution

density.

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The Fourier Transform The Fourier Transform

and and

The Inverse Fourier Transform The Inverse Fourier Transform

 

 

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Are We Finished?

 No! No!

 1 1

^st^st

: We still need to determine the atomic construction (all we : We still need to determine the atomic construction (all we have is electron distribution).

have is electron distribution).

 2 2

^nd^nd

: There are problems with this analysis: : There are problems with this analysis:

 The phase problem The phase problem

 Resolution problems Resolution problems

 Solved with Fitting and Refinement Solved with Fitting and Refinement

 

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Structural Basis for Partial Agonist Action at Structural Basis for Partial Agonist Action at

Ionotropic Glutamate Receptors Ionotropic Glutamate Receptors

 How do partial agonists produce How do partial agonists produce submaximal macroscopic

submaximal macroscopic currents?

currents?

 What is being investigated? What is being investigated?

 GluR2 ligand binding core.GluR2 ligand binding core.

 Why is it being investigated? Why is it being investigated?

 Mechanism by which partial Mechanism by which partial agonists produce submaximal agonists produce submaximal responses remains to be

responses remains to be determined.

determined.

 What is going to be done? What is going to be done?

 4 5-‘R’-willardiines will be used as 4 5-‘R’-willardiines will be used as partial agonists to determine the partial agonists to determine the structure associated with the structure associated with the function.

function.

 Voltage clampingVoltage clamping

 X-ray crystallographyX-ray crystallography

 Outside out membrane patches Outside out membrane patches for single channel analysis

for single channel analysis

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Current Response Current Response

 1 1

^st^st

experiment: experiment:

 Dose Response Analysis using a Dose Response Analysis using a two-electrode voltage clamp on an two-electrode voltage clamp on an oocyte expressing the GluR2

oocyte expressing the GluR2 receptor.

receptor.

 a.) and b.) show affinity of a.) and b.) show affinity of willardiines

willardiines

 Electronegativity is importantElectronegativity is important

 c.) and d.) show that: c.) and d.) show that:

Size does Matter!

 Note relative peak current Note relative peak current amplitude with CTZ:

amplitude with CTZ:

 II_Glu_Glu> I> I_HW_HW> I> I_FW_FW> I> I_BrW_BrW> I> I_IW_IW

 Note steady-state current Note steady-state current amplitude without CTZ:

amplitude without CTZ:

 II_IW_IW > I > I_BrW_BrW> I> I_FW_FW> I> I_Glu_Glu> I> I_HW_HW

 These data suggests that the These data suggests that the efficacy of the XW to

efficacy of the XW to

activate/desensitize the receptor is activate/desensitize the receptor is based on size.

based on size.

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Structure Meets Function Structure Meets Function

 Mode of binding Mode of binding appears similar to appears similar to

glutamate glutamate

 However, the uracil However, the uracil ring and the X

ring and the X

produce a crucial produce a crucial

structural change in structural change in

the ligand-binding the ligand-binding

pocket.

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 Its all about domain Its all about domain closure.

closure.

 Hypothesis: Hypothesis:

 the domains I and II the domains I and II need to be closer to need to be closer to produce an opening of produce an opening of ΔPro632 Δ Pro632

 This opening increases This opening increases ion conductance.

ion conductance.

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Single Channel Analysis Single Channel Analysis

 They ask the question: They ask the question:

 Do receptors populate the same Do receptors populate the same set of subconductance states as set of subconductance states as with full agonists, but have

with full agonists, but have different relative frequencies or different relative frequencies or open times?

open times?

 To Answer the question, they first To Answer the question, they first performed a fluctuation analysis of performed a fluctuation analysis of the macroscopic current by

the macroscopic current by

 slowly applying maximally slowly applying maximally

effective concentrations of Glu, effective concentrations of Glu, IW, and HW on outside-out IW, and HW on outside-out membrane patches.

membrane patches.

 The weighted average The weighted average

conductance with Glu, HW, and conductance with Glu, HW, and IW are 13.1, 11.6, and 7.2 pS.

IW are 13.1, 11.6, and 7.2 pS.

 Suggests that the reduced efficacy Suggests that the reduced efficacy reflects the activation of the open reflects the activation of the open states with different average states with different average conductance.

conductance.

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Amplitude and Duration of Open Amplitude and Duration of Open

States States

 To determine the To determine the

amplitude and duration of amplitude and duration of

the open states, a single the open states, a single

channel analysis of the channel analysis of the steady state responses steady state responses

was carried out.

 Note in a and b, the Note in a and b, the

distributions are the same distributions are the same

(same conductane), so it (same conductane), so it

must be that the open must be that the open

times of the pore for the times of the pore for the

different ligands are different ligands are

different.

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Towards a Structural View of Towards a Structural View of Gating in Potassium Channels Gating in Potassium Channels



Ion Channel has 3 crucial elements:Ion Channel has 3 crucial elements:

 Ion conduction poreIon conduction pore

 Ion gateIon gate

 Voltage sensorVoltage sensor



Architecture of KArchitecture of K_v_v channels channels

 Channel is a tetramerChannel is a tetramer

 N-terminus of S1 is thought to function N-terminus of S1 is thought to function as an intracellular blocker of the pore, as an intracellular blocker of the pore, which underlies fast inactivation—

which underlies fast inactivation—

implies it is inside the membrane implies it is inside the membrane

 S1-S2 linker glycosylated—outside of S1-S2 linker glycosylated—outside of membrane.

membrane.

 S2-S3 cystein can be modified by S2-S3 cystein can be modified by MTS.MTS.

 S3—protein toxins indicate that this is S3—protein toxins indicate that this is close to outside.

close to outside.

 S4 N-terminus is accessible to MTS S4 N-terminus is accessible to MTS outside.

outside.

 S4 & S4-S4 reacts to MTS inside.S4 & S4-S4 reacts to MTS inside.

 S5-S6 is best defined because it S5-S6 is best defined because it remains well conserved across remains well conserved across different channels.

different channels.

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Gate Structure Gate Structure

 Pore domain is formed by S5 and Pore domain is formed by S5 and S6 with S5-S6 lining the pore.

S6 with S5-S6 lining the pore.

 KcsAKcsA

 x-ray structures support this x-ray structures support this model.

model.

 QA—pore blocker—gets stuck QA—pore blocker—gets stuck with rapid hyperpolarization—gate with rapid hyperpolarization—gate is on inside.

is on inside.

 Further experiments indicate that Further experiments indicate that the gate is on the inside.

the gate is on the inside.

 MthKMthK

 Caught in an open state.Caught in an open state.

 Pore Domains Structure and Pore Domains Structure and function

function

 PVP motif (in many channels)—PVP motif (in many channels)—

proline tends to kink helicies.

 Increased MTS reactivity implies a Increased MTS reactivity implies a larger opening with the PVP.

larger opening with the PVP.

 Metal interations not possible in Metal interations not possible in the KcsA or MthK models.

the KcsA or MthK models.

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Voltage Sensors:

The The

Competing Models Competing Models

 S4 region is believed to be the S4 region is believed to be the sensor (charge rich region)

sensor (charge rich region)

 S2 & S3 have been shown to S2 & S3 have been shown to affect the voltage activation affect the voltage activation

relationship.

 Membrane Translocation Membrane Translocation Model

Model

 Protein charges move large Protein charges move large distances through the

distances through the membran.

membran.

 Focused Field Model Focused Field Model

 Protein charges move smaller Protein charges move smaller distances and focus electric distances and focus electric

field across membrane.

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Model Verification!

Or is it?



Note location of S4Note location of S4

 MT Model=yeah!MT Model=yeah!

 FF Model=awwh!FF Model=awwh!



Some ProblemosSome Problemos

 Possible distortions in x-ray Possible distortions in x-ray structure of KvAP

structure of KvAP

 Open and closed structure mixed?Open and closed structure mixed?

 S1-S2 linkers suppose to be S1-S2 linkers suppose to be extracellular—from glycosylation extracellular—from glycosylation sites experiments.

sites experiments.

 A number of other problemsA number of other problems

 PackingPacking

 MTS reactivity on both sides of MTS reactivity on both sides of membrane with approx. the same membrane with approx. the same accessibility, active or not

accessibility, active or not

 Inconsistencies with orientations of Inconsistencies with orientations of other SX components in the other SX components in the structure.

structure.

 Electron Microscopy shows a more Electron Microscopy shows a more expected conformation for the open expected conformation for the open position

position

 Most noted discrepancy is that the Most noted discrepancy is that the N-terminus of S4 and S3 are N-terminus of S4 and S3 are

probably much closer than what the probably much closer than what the x-ray structure shows.

x-ray structure shows.

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Finally:

Evidence for the Models Evidence for the Models

 MTM: MTM:

 Fab Fragments show Fab Fragments show

biotin-avidin complexes on biotin-avidin complexes on

both sides of the both sides of the

membrane. Voltage membrane. Voltage

sensor paddle (S3b-S4) sensor paddle (S3b-S4)

 Red=external Red=external

 Dark blue=internal Dark blue=internal

 Yellow=both Yellow=both

 FFM: FFM:

 Fluorophore attatched to Fluorophore attatched to the N-terminal end of S4 the N-terminal end of S4 maintains its wavelength maintains its wavelength

 Energetically more Energetically more favorable

favorable

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Conclusion Conclusion

 Presented fundamentals of x-ray crystallography Presented fundamentals of x-ray crystallography and how to interpret the data.

and how to interpret the data.

 Presented a paper which discussed structure Presented a paper which discussed structure and function using x-ray crystallography with and function using x-ray crystallography with

GluR2 receptors, and GluR2 receptors, and

 Discussed another paper that reviewed the Discussed another paper that reviewed the current accepted structures of K

current accepted structures of K

_v_v

receptors and receptors and problems/inconsistencies with them.

problems/inconsistencies with them.