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 Post Docs: Andy Ball Ying Lin Stephane Canaan Faculty: Snorri Sigurdsson Michael Gelb Kate Pratt Supported by NSF and NIH

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

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 Distance of AT sequences from probe 

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)?

Move the Neutral Patch Away From the Label

Close Up of High Field Lines

MPs Are More Flexible

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.

Sequences Of Duplex DNA

Sequences Of Duplex DNA cont’d

Goodness of Fit

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

The Goodness of Fit Using Different Models

Flexibility: Force Constant Ratios for different numbers of 50-mer DNAs

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 The trans activation responsive or TAR RNA of HIV interacts with the Tat protein to form the core of the positive transcription elongation factor complex which promotes efficient transcription of the viral genome. In the absence of this interaction, full length viral transcripts are not produced, making this an important site to study as a target for anti-retroviral therapy. The secondary structure of the TAR RNA is shown [next slide] here. PNAS 1998, 95, 12379

Preparation of Spin-Labeled RNA DMTO N NH P CN H C F 3 RNA synthesis RNA deprotection NH2 - RNA O N NH P - RNA H NH2 NCO C l 3 Edwards, T. E., et. al. J. Am. Chem. Soc. 2001, 123, 1527-28 We have developed a method for the site-specific incorporation of nitroxide spin-labels into internal base pairing sites within RNA. This method uses the 2’-amino group as a molecular handle that will react selectively with our spin-labeling reagent, [animation] 4-isocyanto TEMPO, to produce 2’-urea-linked spin-labeled RNAs in high yield. So now that you know how we have done our spin-labeling, let me tell you a little about the RNA that we have been studying.

EPR Spectra of Spin-Labeled TAR RNAs G C A U 5' 3' 40 23 25 38 The RNA contains an upper and lower duplex spanned by a trinucleotide bulge to which the Tat protein binds. We prepared four TAR RNAs, each containing a single spin-label at one of these sites and obtained their EPR spectra. [animation] The spectral width, which goes from the crest of the low field peak to the trough of the high field peak, is a measure of the mobility of the spin-label. The faster the motions of the spin probe, the narrower the spectral width. What we see in the EPR spectra of our spin-labeled TAR RNAs is exactly what we expected. The nucleotides in the flexible bulge, U23 and U25, have a narrower spectral width than those in base-pairing regions, U38 and U40, indicating that they are more mobile. This implies that the spin probe is reporting the motions of the nucleotide to which it is attached. We therefore decided to use information about changes in dynamics to probe RNA structure and function.

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 These are the three topics that we have studied, and I will first describe the interaction of metal ions with the TAR RNA.

High-Resolution Structures of TAR RNA The structure of the TAR RNA in the absence of Tat was solved both by solution NMR in Gabriele Varrani’s laboratory and by crystallography in Tom Steitz’s lab. However, the structures were different. In solution the trinucleotide bulge caused a bend between the upper and lower helixes, whereas in the crystal structure the upper and lower helices have stacked co-axially with a kinked trinucleotide bulge. The crystallographers suggested that the observed four specifically bound calcium ions were responsible for the structural rearrangement. They therefore hypothesized that the calcium ions were important to the physiology of the TAR RNA. We postulated that these two RNA structures would have different internal dynamics and that we could use EPR to test this structural hypothesis. I have shown in red the nucleotides that we have spin-labeled.

EPR of TAR RNAs in the Presence of Cations native Ca2+ Na+ G C A U 5' 3' 40 23 25 38 Shown in black are the native EPR spectra, in magenta are the spectra in the presence of calcium ions and in cyan are the spectra in the presence of sodium ions at the same ionic strength. The spectra in the presence of calcium and sodium ions were nearly identical. Another way to display this data is to plot the change in spectral width as a function of spin-label position . Edwards, T. E., et. al. Chem. Biol. 2002, 9(6), in press

EPR Spectra: “Dynamic Signature” This gives us a dynamic signature for the binding molecule or ligand. As you can see the dynamic signatures are almost identical for sodium and calcium with in increase in spectral width, indicating a decrease in mobility for U23, U25 and U38 and a decrease in spectral width, indicating an increase in mobility for U40. The fact that we observe the same pattern and nearly identical magnitudes of change, indicates to us that there is not a calcium-specific structural rearrangement in solution.

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 The second topic we studied was the binding of derivatives of the Tat protein to the TAR RNA.

Structural Requirements for Tat Binding Tat Derived Peptide (wild type): YGRKKRRQRRR Tat Derived Peptide (mutant): YKKKKRKKKKA We studied the binding of these three derivatives of the Tat protein to the TAR RNA. The short wild-type peptide shown here contains the TAR binding region of Tat. In addition, many researchers have studied the binding of the mutant peptide shown here because it has a similar binding affinity as the wild-type peptide. Because only the central arginine 52 appears to be essential for binding, researchers have also studied the binding of argininamide. In fact, an NMR solution structure of the TAR-argininamide complex was solved in Jamie Williamson’s lab. Argininamide: H 2 N O

High-Resolution Structures of TAR RNA In the argininamide bound structure shown on the right, the upper and lower helices have co-axially stacked like in the calcium bound crystal structure, however, the bulge becomes inverted with U25 folding down and U23 flipping up for form a base triple with A27 and U38 of the upper helix. Therefore, we used EPR to test whether argininmide and the two Tat-derived peptides bind in the same manner.

Dynamic Signatures for TAR RNA Binding I have included the metal ion data for comparison. We see that the Tat derivatives all yield one set of dynamic signatures that is different than the set for the metal ions. For the Tat-derivatives we observed an increase in spectral width, indicative of a decrease in mobility, at nucleotides U23, U38 and U40, whereas U25 had a decrease in spectral width, indicative of increased mobility. However, the magnitude of change in the spectral width was quite different for the wild-type peptide at positions U23 and U38. Interestingly, these two nucleotides participate in the base triple formed upon Tat binding. This difference indicates that amino acids present in the wild-type sequence, but not in the mutant sequence are responsible for the additional decrease in mobility. Because the metal ions gave one set of dynamic signatures and the Tat-derivatives gave another, we wanted to know if other molecules which bind differently would also give a different signature. If we could find that then we would have evidence for a correlation between RNA structure and RNA dynamics. Edwards, T. E., et. al. Chem. Biol. 2002, 9(6), in press

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 So we studied the binding of several small molecules to the TAR RNA.

Small Molecule Inhibitors of TAR These compounds are all well-known inhibitors of the TAR-Tat interaction that bind to several sites in the TAR RNA. The multicyclic dyes, Hoechst 33258, DAPI and berenil bind to the cavity created by the trinucleotide bulge. The Novartis compound CGP 40336A which is the best known TAR inhibitor, binds selectively to this base pair with additional stacking interactions down here. Neomycin binds to the minor groove of the lower helix, whereas argininamide binds to the U23-A27-U38 base triple. We also studied guanidino neomycin which was designed to contain the important binding properties of neomycin and the guanidino group of the essential arginine.

Dynamic Signatures for TAR RNA Binding The multicyclic dyes all gave similar dynamic signatures to each other, but distinctively different than the Novartis compound which was in turn different than neomycin which was different than argininamide. Compounds that bind similarly give similar signatures, whereas those that bind differently give different signatures. Guanidino neomycin bound similarly to argininamide rather than neomycin, illustrating that EPR can be used to provide evidence for specific site binding.

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” In conclusion, we have provided evidence for the absence of a calcium-specific change in solution and have shown that in addition to R52 other amino acids in the wild-type sequence are important for specific binding. We have shown that EPR can be used to predict the binding site for molecules of unknown binding. Take together, our data provides evidence for a strong correlation between RNA dynamics and RNA structure.

spin-labeled RNT 1p RNA-protein complex 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 Bee venom phospholipase Oriented on a membrane surface by Site Directed Mutagenesis EPR spin relaxant method

Human Secretory Phospholipase sPLA2 A highly charged (+20 residues) lipase

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 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.

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

CW-EPR Saturation Method Measure the Height Plot as a function of field or Incident Power Extract the P2 parameter..

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

sPLA2 CW Curves with Membrane

Direct measurement of Spin-Spin Relaxation Rates Bound to membrane (DTPM) vesicles Bound to Mixed Micelles

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 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 Crox salted off protein by addition of NaCl

Orientation different from that of other model. 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.

Extra Thoughts: Model Spin Label All Four First Harmonic Signals

Model Spin Label: All four second harmonic signals

Model Spin Label: Hyperfine Interaction With Protons and FID