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Three Biological Systems: DNA, RNA, Membrane-binding Proteins. Graduate Students: Tamara Okonogi Robert Nielsen Thomas E. Edwards. Post Docs: Andy Ball Ying Lin Stephane Canaan. Faculty: Snorri Sigurdsson Michael Gelb Kate Pratt.

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three biological systems dna rna membrane binding proteins

Three Biological Systems:DNA, RNA, Membrane-binding Proteins

Graduate Students:

Tamara Okonogi

Robert Nielsen

Thomas E. Edwards

Post Docs:

Andy Ball

Ying Lin

Stephane Canaan

Faculty:

Snorri Sigurdsson

Michael Gelb

Kate Pratt

Using EPR as a probe of the Structure-function relation Dynamics-function relation

Supported by NSF and NIH

biological applications of the spin label method

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

a spin labeled base pair

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.

epr 101

EPR 101

The slower moving the label  the wider the spectral width.

Sorry, we have to look at squiggly lines.

cwepr spectra for sl dnas
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.

flexible at sequences inserted in 50mer duplex dna label at position 6
Flexible AT Sequences Inserted in 50mer Duplex DNA Label at position 6

Distance of AT sequences from probe 

methylphosphonates replace phosphates

Removes the negative charge locally (due to the phosphates).

  • Place a line of 10 MPs in a row (UNB)
  • Place a Patch of 6 MPs together (AP)
Methylphosphonates replace Phosphates

MPs are a “phantom model” for protein binding

MPs cause DNA to bend toward the patch.

Is DNA more flexible (bendable)?

does the dna sequence determine flexibility

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.

models for the dnas flexing

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

conclusions about dna dynamics

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

tar rna and replication of the hiv
TAR RNA and Replication of the HIV

TAR RNA

PNAS 1998, 95, 12379

preparation of spin labeled rna
Preparation of Spin-Labeled RNA

O

O

NH

NH

DMTO

N

O

RNA

O

O

N

O

O

RNA synthesis

H

N

C

F

O

O

3

RNA deprotection

N

O

NH2

P

O

O

NH

P

RNA

-

O

RNA

CN

O

O

O

N

O

O

H

O

H

N

N

O

O

O

O

P

RNA

-

N

N

N

O

O

O

O

C

l

O

C

C

l

3

NH2

NCO

Edwards, T. E., et. al. J. Am. Chem. Soc. 2001, 123, 1527-28

epr spectra of spin labeled tar rnas
EPR Spectra of Spin-Labeled TAR RNAs

3'

5'

G

C

G

38

G

C

A

U

25

U

G

C

C

23

U

40

A

U

G

C

A

U

C

G

C

G

G

C

5'

G

C

3'

C

epr studies of tar rna
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
epr of tar rnas in the presence of cations

3'

5'

G

C

G

38

G

C

A

U

25

U

G

C

C

23

U

40

A

U

G

C

A

U

C

G

C

G

G

C

5'

G

C

3'

C

EPR of TAR RNAs in the Presence of Cations

native

Ca2+

Na+

Edwards, T. E., et. al. Chem. Biol. 2002, 9(6), in press

epr studies of tar rna1
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
structural requirements for tat binding
Structural Requirements for Tat Binding

O

N

H

2

H

N

N

H

2

H

N

2

Argininamide:

N

H

Tat Derived Peptide (wild type):

YGRKKRRQRRR

Tat Derived Peptide (mutant):

YKKKKRKKKKA

dynamic signatures for tar rna binding
Dynamic Signatures for TAR RNA Binding

Edwards, T. E., et. al. Chem. Biol. 2002, 9(6), in press

epr studies of tar rna2
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
conclusions
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
NMR: HSQC

spin-labeled RNT 1p RNA-protein complex

RNT 1p protein

Amino acid effect: green = strong

pink = weak

black = none

RNT 1p RNA

membrane binding proteins

Membrane Binding Proteins

Bee venom phospholipase

Oriented on a membrane surface by

Site Directed Mutagenesis

EPR spin relaxant method

human secretory phospholipase spla2

Human Secretory Phospholipase sPLA2

A highly charged (+20 residues) lipase

spin lattice relaxation and rotational motion of the molecule

Spin Lattice Relaxation and Rotational Motion of the Molecule

How CW spectra change with viscosity

How Relaxation Rate R1 changes with viscosity

labeling spla2 with a spin probe
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

The protein should contain only one cysteine for labeling.

Protein labeled at only one site at a time per experiment.

relaxant method nitroxide spectra depend on concentration of relaxants

Relaxant Method: Nitroxide Spectra depend on concentration of relaxants

Rates are increased by the same amount due to additional relaxing agents (relaxants).

Spin-Spin (T1 or R1 processes)

Spin-Lattice (T2 or R2 processes)

cw epr saturation method

CW-EPR Saturation Method

Measure the Height

Plot as a function of field or Incident Power

Extract the P2 parameter..

obtaining relaxation information
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:
relaxant effects for sl spla2 and salt effects

Relaxant effects for sl-sPLA2and Salt Effects

Spectra for spin labeled sPLA2 as a function of ionic strength of NaCl

direct measurement of spin spin relaxation rates
Direct measurement of Spin-Spin Relaxation Rates

Bound to membrane (DTPM) vesicles

Bound to Mixed Micelles

effect of membrane on crox concentration

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.

spla2 on membrane
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

salt effect
Salt Effect

Crox salted off protein by addition of NaCl

spla2 conclusions

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.