Transcript X-Ray Crystallography and It’s Applications

X-Ray Crystallography
and
It’s Applications
By
Bernard Fendler
and
Brad Groveman
Introduction
 Present basic concepts of protein structure
 Discuss why x-ray crystallography is used to
determine protein structure
 Lead through x-ray diffraction experiments
 And present how to utilize experimental
information to design structural models of
proteins
Introduction to Protein Structure:
“The Crystallographer’s Problem”
 What is the crystallographer’s
problem?: Structural
Determination!
 Structure ~ Function
 Amino acids are strung together
on a carbon chain backbone.
 As a result:
 Can be described by the dihedral
angles, called φ, ψ, and ω angles.
 Ramachandran Plot
 Note: the crystallographer is not
in the business of determining
molecular composition, but
determining structural orientation
of a protein.
Introduction to:
X-Ray Crystallography
 x-rays are used to probe the
protein structure:
 Why are x-rays used?
 λ~Å
 Why are crystals used to do x-ray
diffraction?
 Crystals are used because it helps
amplify the diffraction signal.
 How do the x-rays probe the
crystal?
 x-rays interact with the electrons
surrounding the molecule and
“reflect”. The way they are
reflected will be prescribed by the
orientation of the electronic
distribution.
 What is really being measured?
 Electron Density!!!
Performing X-Ray Crystallography Experiments
aka
“Just Do It”
 Bragg’s Law:
 nλ =2dsin(θ)
 Bragg's Law Applet
 X-Ray Diffraction
apparatus.
Performing X-Ray Diffraction
 Resultant diffraction
pattern from
experimental setup
 Diffraction pattern is
actually a Fourier
Transform of the
electron distribution
density.
The Fourier Transform
and
The Inverse Fourier Transform


Are We Finished?
 No!
 1st: We still need to determine the atomic construction (all we
have is electron distribution).
 2nd: There are problems with this analysis:
 The phase problem
 Resolution problems
 Solved with Fitting and Refinement

Structural Basis for Partial Agonist Action at
Ionotropic Glutamate Receptors
 How do partial agonists produce
submaximal macroscopic
currents?
 What is being investigated?
 GluR2 ligand binding core.
 Why is it being investigated?
 Mechanism by which partial
agonists produce submaximal
responses remains to be
determined.
 What is going to be done?
 4 5-‘R’-willardiines will be used as
partial agonists to determine the
structure associated with the
function.
 Voltage clamping
 X-ray crystallography
 Outside out membrane patches
for single channel analysis
Current Response
 1st experiment:
 Dose Response Analysis using a
two-electrode voltage clamp on an
oocyte expressing the GluR2
receptor.
 a.) and b.) show affinity of
willardiines
 Electronegativity is important
 c.) and d.) show that:
Size does Matter!
 Note relative peak current
amplitude with CTZ:
 IGlu> IHW> IFW> IBrW> IIW
 Note steady-state current
amplitude without CTZ:
 IIW > IBrW> IFW> IGlu> IHW
 These data suggests that the
efficacy of the XW to
activate/desensitize the receptor
is based on size.
Structure Meets Function
 Mode of binding
appears similar to
glutamate
 However, the uracil
ring and the X
produce a crucial
structural change in
the ligand-binding
pocket.
 Its all about domain
closure.
 Hypothesis:
 the domains I and II
need to be closer to
produce an opening of
ΔPro632
 This opening increases
ion conductance.
Single Channel Analysis
 They ask the question:
 Do receptors populate the same
set of subconductance states as
with full agonists, but have
different relative frequencies or
open times?
 To Answer the question, they first
performed a fluctuation analysis of
the macroscopic current by
 slowly applying maximally
effective concentrations of Glu,
IW, and HW on outside-out
membrane patches.
 The weighted average
conductance with Glu, HW, and
IW are 13.1, 11.6, and 7.2 pS.
 Suggests that the reduced
efficacy reflects the activation of
the open states with different
average conductance.
Amplitude and Duration of Open
States
 To determine the
amplitude and duration of
the open states, a single
channel analysis of the
steady state responses
was carried out.
 Note in a and b, the
distributions are the same
(same conductane), so it
must be that the open
times of the pore for the
different ligands are
different.
Towards a Structural View of
Gating in Potassium Channels


Ion Channel has 3 crucial elements:



Ion conduction pore
Ion gate
Voltage sensor
Architecture of Kv channels

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Channel is a tetramer
N-terminus of S1 is thought to function
as an intracellular blocker of the pore,
which underlies fast inactivation—
implies it is inside the membrane
S1-S2 linker glycosylated—outside of
membrane.
S2-S3 cystein can be modified by MTS.
S3—protein toxins indicate that this is
close to outside.
S4 N-terminus is accessible to MTS
outside.
S4 & S4-S4 reacts to MTS inside.
S5-S6 is best defined because it
remains well conserved across different
channels.
Gate Structure
 Pore domain is formed by S5 and
S6 with S5-S6 lining the pore.
 KcsA
 x-ray structures support this
model.
 QA—pore blocker—gets stuck
with rapid hyperpolarization—gate
is on inside.
 Further experiments indicate that
the gate is on the inside.
 MthK
 Caught in an open state.
 Pore Domains Structure and
function
 PVP motif (in many channels)—
proline tends to kink helicies.
 Increased MTS reactivity implies a
larger opening with the PVP.
 Metal interations not possible in
the KcsA or MthK models.
Voltage Sensors:
The
Competing Models
 S4 region is believed to be the
sensor (charge rich region)
 S2 & S3 have been shown to
affect the voltage activation
relationship.
 Membrane Translocation
Model
 Protein charges move large
distances through the
membran.
 Focused Field Model
 Protein charges move smaller
distances and focus electric
field across membrane.
Model Verification!
Or is it?


Note location of S4


MT Model=yeah!
FF Model=awwh!
Some Problemos

Possible distortions in x-ray
structure of KvAP
 Open and closed structure mixed?
 S1-S2 linkers suppose to be
extracellular—from glycosylation
sites experiments.
 A number of other problems



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Packing
MTS reactivity on both sides of
membrane with approx. the same
accessibility, active or not
Inconsistencies with orientations of
other SX components in the
structure.
Electron Microscopy shows a more
expected conformation for the open
position
 Most noted discrepancy is that the
N-terminus of S4 and S3 are
probably much closer than what the
x-ray structure shows.
Finally:
Evidence for the Models
 MTM:
 Fab Fragments show
biotin-avidin complexes on
both sides of the
membrane. Voltage sensor
paddle (S3b-S4)
 Red=external
 Dark blue=internal
 Yellow=both
 FFM:
 Fluorophore attatched to
the N-terminal end of S4
maintains its wavelength
 Energetically more
favorable
Conclusion
 Presented fundamentals of x-ray crystallography
and how to interpret the data.
 Presented a paper which discussed structure
and function using x-ray crystallography with
GluR2 receptors, and
 Discussed another paper that reviewed the
current accepted structures of Kv receptors and
problems/inconsistencies with them.