Thursday, December 19, 2013

Carbon CEST and low Spin-lock Field R1rho

Great paper this week by Zhao, Hansen and Zhang from UNC Chapel Hill (& the U of T).  Here is the citation:

The full title is

Characterizing Slow Chemical Exchange in Nucleic Acids by Carbon CEST and Low Spin-lock Field R1rho NMR Spectroscopy.

by Bo Zhao, Alexander Hansen and Qi Zhang

First of all, I've got to give the Zhao et al. credit for a descriptive title.  This JACs communication introduces a new pulse sequence (2D 13C CEST) useful for characterizing slow exchange in nucleic acids.  Then this experiment is compared and contrasted to other tools for characterizing slow exchange, namely R1rho RD and ZZ-exchange.  So as you can see, the title really sums up the paper.

I'll admit that I really like this paper, so let's jump in and see what it is all about.

This paper describes "a nucleic-acid-optimized 2D 13C CEST experiment."  I discussed the concepts behind the CEST experiment a couple of weeks ago, albeit in a slightly different context (see  The experiment described in this week's paper is essentially a selective 2D HSQCs with a long exchange time (Tex) before t1 during which weak B1 is applied to the 13C channel.  This experiment is repeated and the offset of this B1 is arrayed.  The worked-up data (CEST profile) is a plot of the intensity of a given peak vs. offset.  Note that there are as many CEST profiles as peaks in the HSQC spectrum, in principle.  The authors also repeat at different B1 powers.  If the system does not undergo slow chemical exchange the CEST profiles is just a selective saturation experiment (like presat).  When the offset equals the resonance frequency, the peak disappears.  If the system does undergo slow chemical exchange, the saturation of either peak will impact the intensity of the other.  As a result, there are two dips in the CEST profile.

The authors use their new pulse sequence to characterize the ligand-free state of the fluoride riboswitch.  The structure of the ligand-bound form has been determined using crystallography - it is a pseudoknot.  The ligand-free form is hypothesized to have two stem loops based on the 1D 1H NMR.  There are a few extra peaks in the 1D 1H spectrum of the ligand-free RNA, presumably due to slow exchange with another species.  The unstated hypothesis is that this 2nd species is a pseudoknot identical to the ligand-bound form.
One cool trick employed by the authors is to selectively 13C/15N label guanosines in their RNA.  This selective labeling along with a clever use of shaped pulses "to selectively invert and refocus carbon magnetization of interest and to refocus carbon-carbon couplings from neighboring carbons" isolates two G resonances: H8-C8 (base) and H1'-C1' (sugar).  I have played this trick myself, so I know how cool it is to reduce a complicated RNA to a pretty simple and easy to look at spectrum.  If you study the primary sequence of the fluoride riboswitch  you will find 12 G residues.  If you classify these twelve based on the hypothesized secondary structure of the ligand-free RNA and the measured structure of the ligand-bound RNA you would find that - a) there are 4 G residues (G7, G8, G10 and G39) with different secondary structure in free and bound form; b) there are 8 G residues (G1, G2, G4, G14, G23, G30, G31, G33) with the same secondary structure in free and bound form.  These are probes we can use to monitor the slow exchange between these two states using the CEST experiment.

The authors record 13C CEST profiles at 30 C with Tex = 300 ms. (I wonder how they chose these values!).  There is only one dip in the CEST profile of one of the control probes (G33) with the same secondary structure (and presumably chemical shift) in either the ligand-free or bound form.  There are two distinct dips in the CEST profile of two probes (G8 and G10) that transition from a tetraloop secondary structure to a stem-loop upon ligand binding.

The interpretation of this data is that there is an "invisible" excited state (ES) in slow exchange with the ground state (GS) for G8 and G10 (seen in both base and sugar resonances).  For G33 on the other hand, there is no chemical exchange and, thus, no excited state.

The authors make a throw away comment that made me pause: "except for residues from P2, we observed either asymmetrically broadened intensity dips or more than one intensity dip for all other guanosine residues."  I guess this observation makes sense.  After all P1 is a stem-loop in the ligand-free structure and right in the business end of the pseudoloop in the ligand-bound state.  So although it is helical in both structures, it might have different chemical shifts.  So what I am saying is that my division of the 12 guanosines into two easy categories is an oversimplification, at best, and just plain wrong, at worst.

The authors focus their attention on G8 and G10.  Two-state Bloch-McConnell equations are fit to the CEST profiles to extract the ES chemical shift, the exchange rate constant and equilibrium constant.  For the base resonances, the ES chemical shift equals 134.3 and 133.8 ppm for G8 and G10, respectively, which is ~4 ppm upfield from the GS chemical shift.  The average C8 chemical shift for a G residue in a helix equals 133.47.  The exchange rate constant (k1 + k-1) equals 112 +/- 10 Hz.  The population of ES equals 9.8%.  (By the way, with a population that large, why don't you see ES cross-peaks in the HSQC?)  It is trivial to calculate K = 0.112 and deltaG = 5.5 kJ/mol or 1.3 kcal/mol.  Also it is trivial to calculate k1 = 11.2 Hz and k-1 = 100.8 Hz.  (Remember that these values are at 30 C.)

I think it is important to consider these numbers in context.  Arguably, the most prominent NMR dynamics measurement was published WAY BACK in 2005 by Eisenmesser et al. (Nature 2005 438, 117 -  This publication discusses the protein cyclophilin A (CypA).  CypA is in equilibrium between a major and minor form and the authors use relaxation dispersion NMR to measure K = 0.055, k1 = 60 Hz and k-1 = 1080 Hz.  Man, I thought this work was the bee's knees back in the day!  At any rate, this protein refolds with rate constants ~ one-fold larger than the fluorine riboswitch.     

For another example Wenter et al. (Angew. Chem. Int. Ed. 2005 44, 2600 - followed refolding of a bistable 20-mer using real time NMR.  (This paper is one of all-time favorites).  They determine K = 0.236 , k1 = 0.031 +/- 0.006 Hz and k-1 = 0.131 +/- 0.024 Hz at 25 C.  By the way, to ease comparison with the present paper I've flipped the reaction from the Wenter paper so that the more stable fold is the reactant and the less stable fold is the product.  However you look at it, this bistable hairpin refolds with rate constants THREE ORDERS OF MAGNITUDE LESS than the fluoride riboswitch. 

It kind of stuns me that the Wenter 20-mer has such smaller rate constants!  To unfold, the 20-mer does have to break 6 Watson-Crick base pairs and form four new ones.  (I don't know if these two events happen sequentially or simultaneously).  Returning to the Zhao paper, only three or four Watson-Crick base pairs are broken, but nine new base pairs are formed (if we assume the "invisible" species is a pseudoknot identical to the ligand-bound form).  Additionally, some tertiary elements need to form.  So how does the fluoride riboswitch do it so fast?  (This point seems like a good place to admit that I am reading this paper in ASAP form and do not have access to the SI, so I have no idea how much Mg2+ Zhao et al. uses vis-a-vis Wenter et al.)  I guess I should also turn the question around and ask why does the Wenter 20-mer refold so slowly.  Obviously, there is something with the transition state!

To finish up, Zhao and co-workers end their communication by comparing their novel pulse sequence to two more established techniques, low spin-lock field R1rho RD and ZZ exchange.  The results are essentially identical, though ZZ exchange is a bit dicey.  They conclude "The currently presented 2D 13C CEST experiments ... provide powerful tools to investigate slow chemical exchange.  The robustness of these methods promises a unique opportunity to facilitate atomic understanding of slow conformational interconversion that is essential to many vital nucleic acid functions."

I agree.  I like this communication an awful lot and I want to try CEST next time I deal with slow chemical exchange.