Transcript R O B I N S O N Three Biological Systems: DNA, RNA, Membrane-binding Proteins Using EPR as a probe of the Structure-function relation Dynamics-function relation Graduate Students: Post Docs: Faculty: Tamara Okonogi Andy Ball Snorri Sigurdsson Robert.

R
O
B
I
N
S
O
N
Three Biological Systems:
DNA, RNA, Membrane-binding Proteins
Using EPR as a probe of the Structure-function
relation Dynamics-function relation
Graduate Students:
Post Docs:
Faculty:
Tamara Okonogi
Andy Ball
Snorri Sigurdsson
Robert Nielsen
Ying Lin
Michael Gelb
Thomas E. Edwards
Stephane Canaan
Kate Pratt
Supported by NSF and NIH
R
O
B
I
N
S
O
N
Biological Applications of
the Spin Label Method
Bending (Dynamics) of native DNA
polymorphic nature of DNA’s motions
Response of the TAR (to binding proteins)
Structural (and dynamic) response of RNA
Membrane-Binding Proteins
Relation of active site to membrane surface
Comments on EPR’s future
Time Domain, Low Field, High Field
R
O
B
I
N
S
O
N
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.
R
O
B
I
N
S
O
N
EPR 101
The slower moving the label  the wider the
spectral width.
Sorry, we have to look at squiggly lines.
R
O
B
I
N
S
O
N
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.
R
O
B
I
N
S
O
N
Flexible AT Sequences Inserted in
50mer Duplex DNA
Label at position 6
Distance of AT sequences from probe 
R
O
B
I
N
S
O
N
Methylphosphonates replace Phosphates
MPs are a “phantom model” for protein binding
•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)?
R
O
B
I
N
S
O
N
Move the Neutral Patch Away From
the Label
R
O
B
I
N
S
O
N
Close Up of High Field Lines
R
O
B
I
N
S
O
N
MPs Are More Flexible
R
O
B
I
N
S
O
N
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.
R
O
B
I
N
S
O
N
Sequences Of Duplex DNA
Number, Name
L
N1
N2
---
 62   
---
exp

 62   


exp
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
2
6

mod

rad2
R
O
B
I
N
S
O
N
Sequences Of Duplex DNA cont’d
Number, Name
L
N1
N2
 62   
exp

 62   


2
6
exp
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
39. G3C3-motif/A5-tract
G3C3
A5-tract
G3C3
A5-tract
40. A5-tract/G3C3-motif
A5-tract
G3C3
A5-tract
G3C3
6
6
12
22
17
27
0.088 ± 0.001(0.083)
6
5
6
5
2
18
23
29
17
22
28
33
5
6
5
6
12
17
23
28
16
22
27
33
0.089 ± 0.001(0.086)
0.087 ± 0.001(0.072)

mod

rad2
R
O
B
I
N
S
O
N
Goodness of Fit
R
O
B
I
N
S
O
N
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 G
Pyr = T or C
R
O
B
I
N
S
O
N
The Goodness of Fit Using Different Models
R
O
B
I
N
S
O
N
Flexibility: Force Constant Ratios
for different numbers of 50-mer DNAs
R
O
B
I
N
S
O
N
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
R
O
B
I
N
S
O
N
TAR RNA and Replication of the HIV
TAR RNA
PNAS 1998, 95, 12379
R
O
B
I
N
S
O
N
Preparation of Spin-Labeled RNA
O
O
NH
NH
DMTO
N
O
O
RNA
O
O
RNA synthesis
N
O HN
P
O
CF3
O
RNA
CN
O
O
Cl
NH2
O
P
-
O
NH2
NH
RNA
O
O
N
O
O
RNA deprotection
O
N
N
O
O
RNA
O
P
NCO
Edwards, T. E., et. al. J. Am. Chem. Soc. 2001, 123,
1527-28
-
O
O
H
N
O HN
O
OCCl3
N
O
N
O
R
O
B
I
N
S
O
N
EPR Spectra of Spin-Labeled TAR RNAs
3'
G
C
G
A
25U G
C
23U
A
G
A
C
C
G
G
5'
5'
G
C
U 38
C
U 40
C
U
G
G
C
C
C
3'
R
O
B
I
N
S
O
N
EPR Studies of TAR RNA
• Interactions of metal ions with the TAR
RNA
• Binding of Tat-derivatives to the TAR RNA
• Inhibition of the TAR RNA by small
molecules
R
O
B
I
N
S
O
N
High-Resolution Structures of TAR RNA
R
O
B
I
N
S
O
N
EPR of TAR RNAs in the Presence of Cations
3'
G
C
G
A
25U G
C
23U
A
G
A
C
C
G
G
5'
native
Ca2+
Na+
5'
G
C
U 38
C
U 40
C
U
G
G
C
C
C
3'
Edwards, T. E., et. al. Chem. Biol. 2002, 9(6), in
press
R
O
B
I
N
S
O
N
EPR Spectra: “Dynamic Signature”
R
O
B
I
N
S
O
N
EPR Studies of TAR RNA
• Interactions of metal ions with the TAR
RNA
• Binding of Tat-derivatives to the TAR RNA
• Inhibition of the TAR RNA by small
molecules
R
O
B
I
N
S
O
N
Structural Requirements for Tat Binding
Tat Derived Peptide (wild type): YGRKKRRQRRR
Tat Derived Peptide (mutant):
O
Argininamide:
H2 N
YKKKKRKKKKA
NH2
H
N
NH2
NH
R
O
B
I
N
S
O
N
High-Resolution Structures of TAR RNA
R
O
B
I
N
S
O
N
Dynamic Signatures for TAR RNA Binding
Edwards, T. E., et. al. Chem. Biol. 2002, 9(6), in
press
R
O
B
I
N
S
O
N
EPR Studies of TAR RNA
• Interactions of metal ions with the TAR
RNA
• Binding of Tat-derivatives to the TAR RNA
• Inhibition of the TAR RNA by small
molecules
R
O
B
I
N
S
O
N
Small Molecule Inhibitors of TAR
R
O
B
I
N
S
O
N
Dynamic Signatures for TAR RNA Binding
R
O
B
I
N
S
O
N
Conclusions
 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”
NMR: HSQC
R
O
B
I
N
S
O
N
spin-labeled RNT 1p RNA-protein complex
G U*
A
ef f ect
RNT 1p protein
A
C G
A U*
no ef f ect
G C
5'
U*
no ef f ect
U
RNT 1p RNA
Amino acid effect: green = strong
pink = weak
black = none
R
O
B
I
N
S
O
N
Membrane Binding Proteins
Bee venom phospholipase
Oriented on a membrane surface by
Site Directed Mutagenesis
EPR spin relaxant method
R
O
B
I
N
S
O
N
Human Secretory Phospholipase sPLA2
A highly charged (+20 residues) lipase
R
O
B
I
N
S
O
N
Spin Lattice Relaxation
and Rotational Motion of the Molecule
How CW spectra change with viscosity
How Relaxation Rate R1 changes with viscosity
R
O
B
I
N
S
O
N
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
PLA2
C
O
PLA2
+
O
H3C S
SH
S
C
S
S
CH2
CH2
N
N
.O
.O
The protein should contain only one cysteine for labeling.
Protein labeled at only one site at a time per experiment.
R
O
B
I
N
S
O
N
Relaxant Method:
Nitroxide Spectra depend on concentration of relaxants
R1  R1o     Rlxnt 
Spin-Spin (T1 or R1 processes)
R2  R     Rlxnt 
Spin-Lattice (T2 or R2 processes)
o
2
Rates are increased by the same amount due to
additional relaxing agents (relaxants).
P2  R1 R2  R     Rlxnt R     Rlxnt 
o
1
R  R
o
2
o
1
o
2
R     Rlxnt 
o
2
P2  P2  P  R  R
0
2
o
1
o
2
    Rlxnt 
R
O
B
I
N
S
O
N
CW-EPR Saturation Method
• Measure the Height
• Plot as a function of field or Incident Power
• Extract the P2 parameter..
R
O
B
I
N
S
O
N
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:
Y 
1
2
cPo
 Po 
1  
 P2 

1
2
 
3
2
R
O
B
I
N
S
O
N
Relaxant effects for sl-sPLA2
and Salt Effects
Spectra for spin labeled sPLA2 as a function of
ionic strength of NaCl
R
O
B
I
N
S
O
N
sPLA2 CW Curves with Membrane
R
O
B
I
N
S
O
N
Direct measurement of Spin-Spin
Relaxation Rates
Bound to membrane (DTPM) vesicles
Bound to Mixed Micelles
R
O
B
I
N
S
O
N
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.
 membrane
1
no  membrane
1
R

R
Crox 


no  membrane
Crox
 membrane
R
O
B
I
N
S
O
N
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
R
O
B
I
N
S
O
N
Salt Effect
Crox salted off protein by addition of NaCl
R
O
B
I
N
S
O
N
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.
R
O
B
I
N
S
O
N
Extra Thoughts: Model Spin Label
All Four First Harmonic Signals
R
O
B
I
N
S
O
N
Model Spin Label:
All four second harmonic signals
R
O
B
I
N
S
O
N
Model Spin Label:
Hyperfine Interaction With Protons and
FID