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

1. 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

2. 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

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. 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)?

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 cont’d

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 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

16. The Goodness of Fit Using Different Models

17. Flexibility: Force Constant Ratiosfor 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 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

21. 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

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. 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

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 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

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 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)

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