![Page 1: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/1.jpg)
ROBINSON
Three Biological Systems:DNA, RNA, Membrane-binding
Proteins
Using EPR as a probe of the Structure-function relation Dynamics-function
relation
Graduate Students:
Tamara Okonogi
Robert Nielsen
Thomas E. Edwards
Faculty:
Snorri Sigurdsson
Michael Gelb
Kate Pratt
Post Docs:
Andy Ball
Ying Lin
Stephane Canaan
Supported by NSF and NIH
![Page 2: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/2.jpg)
ROBINSON
Biological Applications of the Spin Label Method
Bending (Dynamics) of native DNApolymorphic nature of DNA’s motions
Response of the TAR (to binding proteins)Structural (and dynamic) response of
RNA
Membrane-Binding ProteinsRelation of active site to membrane
surface
Comments on EPR’s futureTime Domain, Low Field, High Field
![Page 3: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/3.jpg)
ROBINSON
A Spin Labeled Base Pair
Replace a natural base pair with a spin labeled one.
Using phosphoramadite chemistry, construct DNAs of any length and sequence.
Make the duplex from xs complement.
![Page 4: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/4.jpg)
ROBINSON
EPR 101
The slower moving the label the wider the spectral width.
Sorry, we have to look at squiggly lines.
![Page 5: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/5.jpg)
ROBINSON
CWEPR Spectra for sl-DNAs
Two different isotopes of spin labels. For duplex DNAs of different lengths, with the spin label uniquely in the middle of each DNA.
![Page 6: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/6.jpg)
ROBINSON
Flexible AT Sequences Inserted in 50mer Duplex DNA
Label at position 6
Distance of AT sequences from probe
![Page 7: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/7.jpg)
ROBINSON
Methylphosphonates replace Phosphates
•Place a line of 10 MPs in a row (UNB)
•Place a Patch of 6 MPs together (AP)
Removes the negative charge locally (due to the phosphates).
MPs cause DNA to bend toward the patch.
Is DNA more flexible (bendable)?
MPs are a “phantom model” for protein binding
![Page 8: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/8.jpg)
ROBINSON
Move the Neutral Patch Away From the Label
![Page 9: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/9.jpg)
ROBINSON
Close Up of High Field Lines
![Page 10: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/10.jpg)
ROBINSON
MPs Are More Flexible
![Page 11: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/11.jpg)
ROBINSON
Does the DNA sequence determine flexibility?
•We examined many (40) different sequences.
•Measured the dynamics for each sequence
•All duplex DNAs were 50 base pairs long
•All duplex DNAs had the first 12 base pairs constant
•The probe was always at postion 6.
As a sequence is moved further from the duplex DNA its effect falls off.
![Page 12: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/12.jpg)
ROBINSON
Sequences Of Duplex DNA
1. NT 0 --- --- 0.087 ± 0.002
2. AT4 8 12 19 0.090 ± 0.001
3. AT10 20 12 31 0.093 ± 0.001
4. AT15 30 12 41 0.097 ± 0.003
5. AT7A 15 12 26 0.093 ± 0.001
6. AT7A_s5 15 17 31 0.091 ± 0.001
7. AT7A_s12 15 24 38 0.088 ± 0.001
8. AT7A_s24 15 36 50 0.087 ± 0.002
9. AA7A or AAA5 15 12 26 0.083 ± 0.001
10.AA10 20 12 31 0.089 ± 0.001(0.084)
11.AA7A_s5 15 17 31 0.086 ± 0.001(0.084)
12.CG7C 15 12 26 0.085 ± 0.002
13.CG10 20 12 31 0.086 ± 0.001
14.CG7C_s5 15 17 31 0.086 ± 0.001
15.CC7C or CCC5 15 12 26 0.086 ± 0.002
16CC10 20 12 31 0.084 ± 0.001
17.CC7C_s5 15 17 31 0.087 ± 0.001
18.AC7A 15 12 26 0.088 ± 0.002
19.AG7A 15 12 26 0.089 ± 0.002
20.AAT5 15 12 26 0.089 ± 0.004
21.AAC5 15 12 26 0.088 ± 0.001
22.AAC5_s5 15 17 31 0.087 ± 0.001
23.AAG5 15 12 26 0.087 ± 0.002
24.AGG5 15 12 26 0.089 ± 0.001
25.AGG5_s5 15 17 31 0.087 ± 0.001
26.ACG5 15 12 26 0.089 ± 0.001
Number, Name L N1 N2
2
6 exp 26
exp
2
6 mod
rad2
![Page 13: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/13.jpg)
ROBINSON
Sequences Of Duplex DNA cont’d
27.ACG5_s5 15 17 31 0.085 ± 0.001
28.ACT5 15 12 26 0.088 ± 0.001
29.ATC5 15 12 26 0.087 ± 0.002
30.CAG5 15 12 26 0.089 ± 0.002
31.CCG5 15 12 26 0.087 ± 0.001
32.CCG5_s5 15 17 31 0.082 ± 0.002
33.1/2CAP: TGTGACAT 8 12 19 0.089 ± 0.002
34.TATA: TATATAAA 8 12 19 0.093 ± 0.002(0.088)
35.G3C3-motif 6 12 17 0.084 ± 0.001(0.083)
36.G3C3-motif_s1 6 13 18 0.081 ± 0.001(0.077)
37.G3C3-motif_s8 6 20 25 0.076 ± 0.001(0.070)
38.G3C3-motif_s0_s10 G3C3-motif_s10
6 6
12 17 22 27
0.088 ± 0.001(0.083)
39. G3C3-motif/A5-tract G3C3 A5-tract G3C3 A5-tract
6 5 6 5
2 17 18 22 23 28 29 33
0.089 ± 0.001(0.086)
40. A5-tract/G3C3-motif A5-tract G3C3 A5-tract G3C3
5 6 5 6
12 16 17 22 23 27 28 33
0.087 ± 0.001(0.072)
Number, Name L N1 N2
2
6 exp 26
exp
2
6 mod
rad2
![Page 14: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/14.jpg)
ROBINSON
Goodness of Fit
![Page 15: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/15.jpg)
ROBINSON
Models for the DNAs flexing
Considered 3 different types of flexibility in A Nearest Neighbor picture (a di-nucleotide model)
• 3 parameters: pur-pur (same as pyr-pyr), pur-pyr, and pyr-pur are the three distinct steps
• 6 parameters: AT is different from GC and order doesn’t matter. (Hogan-Austin Model)
• 10 Parameter: All dinucleotide steps are unique (the two stiffest were so stiff we had to fix them)
Pur = A or GPyr = T or C
![Page 16: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/16.jpg)
ROBINSON
The Goodness of Fit Using Different Models
![Page 17: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/17.jpg)
ROBINSON
Flexibility: Force Constant Ratios for different numbers of 50-mer DNAs
![Page 18: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/18.jpg)
ROBINSON
Conclusions about DNA dynamics
DNA (measured by EPR, fast time-scale) is three times stiffer than that measured by traditional methods:
Demonstrate polymorphic nature of duplex DNA and suggests the existence of slowly relaxing structures.
Certain sequences are inherently more flexible.
Eg: AT runs and charge neutral (MP) sequences.
Sequence dependent DNA flexibility does not discriminate between AT vs GC (regardless of order).
The Hogan-Austin hypothesis is wrong.
Sequence does discriminate between purines and pyrimidines.
The step from (5’) CG to a GC (3’) is most flexible (CpG step)
The step from (5’) CG to a GC (3’) is most flexible
The step from (5’) TA to a AT (3’) is next-most flexible
![Page 19: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/19.jpg)
ROBINSON
TAR RNA
PNAS 1998, 95, 12379
TAR RNA and Replication of the HIV
![Page 20: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/20.jpg)
ROBINSON
ODMTO N
NH
O
O
O
PN
OCN
HN CF3
O
RNA synthesis
RNA deprotection
OO N
NH
O
O
O
PO
OO
NH2
-RNA
RNA
OO N
NH
O
O
O
PO
OO -RNA
RNA
O
HNHN
NO
N
O
NH2
N
O
NCO
Cl OCCl3
O
Edwards, T. E., et. al. J. Am. Chem. Soc. 2001, 123, 1527-28
Preparation of Spin-Labeled RNA
![Page 21: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/21.jpg)
ROBINSON
GCGAG
AGACCGG
GCUC
UCUGGCCC5'
3'
5'3'
C
40U23
U25
38
EPR Spectra of Spin-Labeled TAR RNAs
![Page 22: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/22.jpg)
ROBINSON
• Interactions of metal ions with the TAR RNA
• Binding of Tat-derivatives to the TAR RNA
• Inhibition of the TAR RNA by small molecules
EPR Studies of TAR RNA
![Page 23: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/23.jpg)
ROBINSON
High-Resolution Structures of TAR RNA
![Page 24: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/24.jpg)
ROBINSON nativ
eCa2+
Na+
GCGAG
AGACCGG
GCUC
UCUGGCCC5'
3'
5'3'
C
40U23
U25
38
Edwards, T. E., et. al. Chem. Biol. 2002, 9(6), in press
EPR of TAR RNAs in the Presence of Cations
![Page 25: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/25.jpg)
ROBINSON
EPR Spectra: “Dynamic Signature”
![Page 26: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/26.jpg)
ROBINSON
• Interactions of metal ions with the TAR RNA
• Binding of Tat-derivatives to the TAR RNA
• Inhibition of the TAR RNA by small molecules
EPR Studies of TAR RNA
![Page 27: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/27.jpg)
ROBINSON
Argininamide:H2N
NH2O
HN NH2
NH
Tat Derived Peptide (mutant): YKKKKRKKKKA
Tat Derived Peptide (wild type): YGRKKRRQRRR
Structural Requirements for Tat Binding
![Page 28: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/28.jpg)
ROBINSON
High-Resolution Structures of TAR RNA
![Page 29: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/29.jpg)
ROBINSON
Edwards, T. E., et. al. Chem. Biol. 2002, 9(6), in press
Dynamic Signatures for TAR RNA Binding
![Page 30: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/30.jpg)
ROBINSON
• Interactions of metal ions with the TAR RNA
• Binding of Tat-derivatives to the TAR RNA
• Inhibition of the TAR RNA by small molecules
EPR Studies of TAR RNA
![Page 31: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/31.jpg)
ROBINSON
Small Molecule Inhibitors of TAR
![Page 32: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/32.jpg)
ROBINSON
Dynamic Signatures for TAR RNA Binding
![Page 33: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/33.jpg)
ROBINSON
No calcium-specific change, as suggested by crystallography, was observed in solution by EPR
The wild-type Tat peptide causes a dramatic decrease in the motion of U23 and U38, implying that in addition to R52 other amino acids are important for specific binding
EPR can predict specific site binding
Taken together, our results provide evidence for a strong correlation between RNA-protein interactions and RNA “dynamic signature”
Conclusions
![Page 34: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/34.jpg)
ROBINSON
Amino acid effect: green = strong pink = weak black = none
spin-labeled RNT 1p RNA-protein complex
AG U*
A
C
A
G
U*
G
C
U*U
no effect
no effect
effect
5'
RNT 1p RNA
RNT 1p protein
NMR: HSQC
![Page 35: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/35.jpg)
ROBINSON
Membrane Binding Proteins
Bee venom phospholipase
Oriented on a membrane surface by
Site Directed Mutagenesis
EPR spin relaxant method
![Page 36: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/36.jpg)
ROBINSON
Human Secretory Phospholipase sPLA2
A highly charged (+20 residues) lipase
![Page 37: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/37.jpg)
ROBINSON
Spin Lattice Relaxation and Rotational Motion of the
Molecule How CW spectra change with viscosity
How Relaxation Rate R1 changes with viscosity
![Page 38: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/38.jpg)
ROBINSON
Labeling sPLA2 with a Spin Probe
Use site directed mutagenesis techniques to prepare proteins with a single
properly placed cytsteine. General Reaction for adding relaxants
H3C S S CH2
N OO
OS CH2
N O
PLA2 C SHPLA2 C S
+
..
The protein should contain only one cysteine for labeling.
Protein labeled at only one site at a time per experiment.
![Page 39: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/39.jpg)
ROBINSON
Relaxant Method: Nitroxide Spectra depend on concentration of
relaxants
Spin-Spin (T1 or R1 processes)
Spin-Lattice (T2 or R2 processes)
1 1
2 2
o
o
R R Rlxnt
R R Rlxnt
Rates are increased by the same amount due to additional relaxing agents (relaxants).
2 1 2 1 2
2 1 2
02 2 2 1 2
o o
o o o
o o
P R R R Rlxnt R Rlxnt
R R R Rlxnt
P P P R R Rlxnt
![Page 40: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/40.jpg)
ROBINSON
CW-EPR Saturation Method
• Measure the Height
• Plot as a function of field or Incident Power
• Extract the P2 parameter..
![Page 41: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/41.jpg)
ROBINSON
Obtaining Relaxation Information
• Time Domain (Saturation Recovery or Pulsed ELDOR) depends on R1, directly.
• CW method (progressive saturation or rollover”) depends on P2.
• Signal Height is a function of incident microwave power:
12
2
1
o
o
cPY
P
P
312 2
![Page 42: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/42.jpg)
ROBINSON
Relaxant effects for sl-sPLA2and Salt Effects
Spectra for spin labeled sPLA2 as a function of ionic strength of NaCl
![Page 43: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/43.jpg)
ROBINSON
sPLA2 CW Curves with Membrane
![Page 44: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/44.jpg)
ROBINSON
Direct measurement of Spin-Spin Relaxation Rates
Bound to membrane (DTPM) vesicles
Bound to Mixed Micelles
![Page 45: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/45.jpg)
ROBINSON
Effect of Membrane on Crox Concentration
Exposure factor as a function of distance from the membrane surface. Crox is z=-3 and the membrane is negatively charged.
1
1
membranemembrane
no membraneno membrane
CroxR
R Crox
![Page 46: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/46.jpg)
ROBINSON
sPLA2 on Membrane
View from membrane
Yellow: Hydrophobic Residues
Blue: Charged (pos) residues
Orientation perpendicular to that predicted by M. Jain.
Anchored by hydrophobic residues. Charges not essential
![Page 47: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/47.jpg)
ROBINSON
Salt EffectCrox salted off protein by addition of NaCl
![Page 48: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/48.jpg)
ROBINSON
sPLA2 Conclusions
sPLA2 causes the vesicles to aggregate.
Explains much other data and misconceptions about the kinetics and processive nature of sPLA2 action.
sPLA2 was oriented on micelles (instead) using spin-spin relaxation rates alone.
Orientation different from that of other model.
Hydrophobic residues are the main points of contact.
Charges provide a general, non-specific attraction.
![Page 49: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/49.jpg)
ROBINSON
Extra Thoughts: Model Spin Label All Four First Harmonic Signals
![Page 50: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/50.jpg)
ROBINSON
Model Spin Label: All four second harmonic signals
![Page 51: ROBINSONROBINSON Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function](https://reader036.vdocuments.us/reader036/viewer/2022062516/56649d545503460f94a31c67/html5/thumbnails/51.jpg)
ROBINSON
Model Spin Label:Hyperfine Interaction With Protons
and FID