Tools of the Trade
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Tools of the Trade 1- Atomic resolution: X-ray crystallography 2- NMR spectroscopy 3- de novo Modeling and structure determination, Homology modeling 4- CryoEM Why do we needs probes ????? Issues: 1- sample requirement 2- nature of the environment 3- Dynamics and time scales
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Tools of the Trade. 1 - Atomic resolution: X-ray crystallography 2 - NMR spectroscopy 3 - de novo Modeling and structure determination, Homology modeling 4 - CryoEM Why do we needs probes ????? Issues: 1- sample requirement 2- nature of the environment 3- Dynamics and time scales. - PowerPoint PPT Presentation
Transcript of Tools of the Trade
Tools of the Trade
1- Atomic resolution: X-ray crystallography
2- NMR spectroscopy
3- de novo Modeling and structure determination, Homology modeling
4- CryoEM
Why do we needs probes ?????
Issues: 1- sample requirement2- nature of the environment3- Dynamics and time scales
Functional Dynamics of Proteins
NO
CH2 S S CH3
O
OMethanethiosulfonate
spin label
Site-directed mutagenesis
S
C NHCH
CNH
CHC
O
O
O
NH3+
C NHCH
CNH
CHC
O
O
O
NH3+SH
C NHCH
CNH
CHC
O
O
O
NH3+
S
N O
Side Chain R1
Site-Directed Spin Labeling
Spectral parameter Protein structure
Probe spectroscopy
Biggest challenge: What is the transfer function?Examples :
Uncertainty principle of chemical modification:Implications: limited resolution both on the static and dynamic levels
Functional analysis of labeled mutants is a REQUIREMENT
Electron Paramagnetic ResonanceElectron Spin Resonance
Mn+2
OH. N
O
R1 R2
.
Free RadicalsAscorbateHydroxylTocopheroletc.
Transition MetalsAll that haveunpaired electrons
Spin LabelsR1 & R2 chosen to provide specificity
Spin ½ Electron in an External Field
Electron Spins Precess@ the Larmor Frequencyabout the External Fielde = 1.76e7 rad/sec/G
Approximate Energy Levels for a 14N Nitroxide Spin Label; S =½, I=1
In general, EPR givesinformation on the #and nuclear spin stateof nearby nuclei –coordination sphere ofmetal centers in in metalloproteins
Energy level Diagram for a 15N NitroxideS=½; I=½
Block Diagram of EPR Spectrometer
SolidsLiquidsGasesHigh S/N
Most EPRdone @X-band9-10 GHz3 kG Field
Nitroxide Reference Frame
Hres = [h/egeff)] + miAeff()
geff = gxxsin2cos2gyysin2sin2 + gzzcos2
Aeff = [Axx2sin2cos2 + Ayy
2sin2sin2 + Azz2cos2] ½
gxx = 2.0086gyy = 2.0056gzz = 2.0022Axx = 7 GAyy = 6 GAzz = 34 G
Variation in the EPR Spectrum for a Single Nitroxide
Single Crystal - x
Single Crystal - y
Single Crystal - z
Polycrystalline
Crystal Dissolvedin Water
Two-Site Exchange ModelEffects of Motion on EPR Spectra
EPR Spectra as a Function of RotationalCorrelation Time; r = 1/6Dr
Nitroxide in H20
Small Protein - EGF
Large Protein – 1 MDa
r = 4rh3/ 3kT
Electron Spin-Spin Relaxation - AccessibilityNitroxide – slow relaxation
T1e’s of isolated Nitroxide spin labels are on the order of afew sec’s at physiological temperatures
Electron Spin-Spin Relaxation - AccessibilityNitroxide – slow relaxation
Nicklel Complexes – fast relaxationOther Transition Metals and O2
