Three Biological Systems: DNA, RNA, Membranebinding Proteins

1
Three Biological SystemsDNA, 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
2
Biological Applications of the Spin Label Method

• Bending (Dynamics) of native DNA
• polymorphic nature of DNAs 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 EPRs future
• Time Domain, Low Field, High Field

3
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.
4
EPR 101

• The slower moving the label ? the wider the
  spectral width.
• Sorry, we have to look at squiggly lines.

5
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.
6
Flexible AT Sequences Inserted in 50mer Duplex
DNA Label at position 6
Distance of AT sequences from probe ?
7
Methylphosphonates replace Phosphates
MPs are a phantom model for protein binding
MPs cause DNA to bend toward the patch. Is DNA
more flexible (bendable)?
8
Move the Neutral Patch Away From the Label
9
Close Up of High Field Lines
10
MPs Are More Flexible
11
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.

12
Sequences Of Duplex DNA
13
Sequences Of Duplex DNA contd
14
Goodness of Fit
15
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
  doesnt 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

16
The Goodness of Fit Using Different Models
17
Flexibility Force Constant Ratios for
different numbers of 50-mer DNAs
18
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

19
TAR RNA and Replication of the HIV
TAR RNA
PNAS 1998, 95, 12379
20
Preparation of Spin-Labeled RNA
21
EPR Spectra of Spin-Labeled TAR RNAs
22
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

23
High-Resolution Structures of TAR RNA
24
EPR of TAR RNAs in the Presence of Cations
native Ca2 Na
Edwards, T. E., et. al. Chem. Biol. 2002, 9(6),
in press
25
EPR Spectra Dynamic Signature
26
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

27
Structural Requirements for Tat Binding
28
High-Resolution Structures of TAR RNA
29
Dynamic Signatures for TAR RNA Binding
Edwards, T. E., et. al. Chem. Biol. 2002, 9(6),
in press
30
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

31
Small Molecule Inhibitors of TAR
32
Dynamic Signatures for TAR RNA Binding
33
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

34
NMR HSQC
spin-labeled RNT 1p RNA-protein complex
RNT 1p protein
Amino acid effect green strong pink
weak black none
RNT 1p RNA
35
Membrane Binding Proteins

• Bee venom phospholipase
• Oriented on a membrane surface by
• Site Directed Mutagenesis
• EPR spin relaxant method

36
Human Secretory Phospholipase sPLA2

• A highly charged (20 residues) lipase

37
Spin Lattice Relaxation and Rotational Motion of
the Molecule

• How CW spectra change with viscosity
• How Relaxation Rate R1 changes with viscosity

38
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.
39
Relaxant Method Nitroxide Spectra depend on
concentration of relaxants

• Spin-Spin (T1 or R1 processes)
• Spin-Lattice (T2 or R2 processes)

Rates are increased by the same amount due to
additional relaxing agents (relaxants).
40
CW-EPR Saturation Method

• Measure the Height
• Plot as a function of field or Incident Power
• Extract the P2 parameter..

41
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

42
Relaxant effects for sl-sPLA2and Salt Effects

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

43
sPLA2 CW Curves with Membrane
44
Direct measurement of Spin-Spin Relaxation Rates
Bound to membrane (DTPM) vesicles
Bound to Mixed Micelles
45
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.

46
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
47
Salt Effect

• Crox salted off protein by addition of NaCl

48
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.

49
Extra Thoughts Model Spin Label All Four First
Harmonic Signals
50
Model Spin Label All four second harmonic
signals
51
Model Spin LabelHyperfine Interaction With
Protons and FID