Finding side chain resonances using TOCSY data

Navigation to Peaks

The next kind of resonance assignment that we will look as is for the assignment of amino acid side chain atoms. As an example we will choose a relatively simple residue, with relatively clear peaks, naturally some residues with obscures or weak peaks will be more difficult to assign than this one, but the general principles remain the same. Firstly we will load a new project with 3D TOCSY spectra loaded. Select to close the exiting project, but do not save, then navigate to find and select the CcpnCourse2b project and lastly click [Open]. This project contains an assigned HSQC spectrum, some tripe resonance backbone (HNC) experiments and three different kinds of three-dimensional TOCSY spectra.

In window2 a three-dimensional CCcoNH TOCSY spectrum is shown. Like the HNcoCA and HNcoCACB spectra, this shows peaks belonging to amide resonances which correlate with the carbon resonances of the previous residue. For example, below  we will look for the 20 Ala H,N (amide) position but the side chain peaks on the Y-axis will be for residue 19 Thr:

In window3 a three-dimensional 15N HSQC TOCSY is shown. This spectrum shows side chain hydrogen resonances on the Y-axis and amide (HSQC) resonances on the other axes. Here the amide resonances are from the same residue as the amides:

In window5 a three-dimensional HCcH TOCSY is shown. This spectrum has two hydrogen axes and one carbon axis. The hydrogen dimension on the X-axis represents resonances that are directly bound to the carbon resonances on the Z-axis (depth axis). This contracts with the other side chain spectra where the Z axis is for amide nitrogen. Thus here we are looking at CH pairs not NH pairs. The Y-axis of this spectrum represents hydrogen resonances that are in the same residue as the bound CH pair on the other axes:

In this example we will assign the side chain peaks for the 19 Thr residue, thus we expect to find threecarbon resonances; CA, CB, & CG2  and three hydrogen resonances; HA, HB, HG2* (methyl). To find the hydrogen and carbon resonances in the amide-linked spectra we will navigate from the HSQC peak positions. Accordingly, open M: Peak: Peak Lists, select the {Peak Table} tab and set the peak list pulldown menu to "HSQC:115:1". Now sort the HSQC peaks by sequence by clicking on one of the "Assign" column headings in the table. Scroll down so that peaks for 19Thr and 20Ala are visible. Select the 20Ala row, then above the table set pulldown menu next to "Go To Position" to "1H - 15N in window2" and click [Go To Position]. This will cause the display of window2 to move to the amide of 20Ala, but because the CCcoNH spectrum shows side chain resonances for the previous residue, the peaks will derive from 19Thr in their middle dimension. Go back to the peak list table and select the row for 19Thr, change the pulldown menu next to "Go To Position" to "1H - 15N in window3" and click [Go To Position]. This will show the amide and side chain peaks for 19Thr in the HSQC TOCSY spectrum.

With window2 and window3 located at the carbon and hydrogen side chain positions for 19Thr we next assign the non-amide (middle) dimension of all the relevant peaks. For the CCcoNH spectrum in window2 place the mouse over the peak labelled as "20Ala H,-,N" near 69 ppm on the Y-axis (carbon):

and press 'a' to open the Assignment: Assignment Panel popup. This is the CB peak for 19Thr, so when the Assignment Panel opens, you will see "19ThrCb" as an assignment option in the middle table. Click on the "19ThrCb" row to assign this to the middle dimension of the peak. Note that this CB resonance exists and has a known chemical shift because if the assignments in the HNcoCACB and HNCACB backbone experiments. Repeat this procedure for the peak at 66 ppm ( press 'a' over it) to give it a "19ThrCa" assignment in its middle dimension:

Assigning New Side Chain Resonances

For the last carbon position at 20 ppm we will make an assignment to the CG2 atom, however this resonance is new to the project, and hence there is no assignment suggestion that appears automatically for the middle dimension. Instead we will add a new resonance that we will link to the right atoms. To do this click on the middle [<New>] button (F2 dimension). You will see a new resonance number appear of the form [number] in the middle dimension:

Click on this new resonance in the left most column, then click [Assign [number]] at the left of the blue buttons. In the Molecule: Atom Browser popup that appears, switch on the hydrogen and carbon atoms using the [C] and [H] buttons. Then scroll down to the 19 Thr residue and click on the right most "Cg2" (the "g" will be a Greek gamma). Returning to the Assignment: Assignment Panel you will now see that the resonance assigned to the middle dimension is labelled as "19ThrCg2":

Next move to window3 and assign the side chain peaks of the HSQC TOCSY spectrum in a similar way. In this case the peaks you need to consider will be initially labelled as "19Thr H,-,N" because the middle dimension will carry the hydrogen assignments for the same residue as the amide. There will be three peaks at 4.26, 3.82 and 1.21 ppm, these correspond to the HB. HA and HG2 resonances respectively. Note that the HB peak is below the HA peak because this is a Thr residue. For each of these peaks you will need to make a new resonance with [<New>] and use [Assign [number]] to link it to the right atoms.

Assigning the HCcH TOCSY

For the  HCcH TOCSY spectrum we will use the location of the side chain resonances we have just assigned in the other TOCSY spectra to locate peak positions. Note that this spectrum does not have any peaks picked in it. The idea is that the CCcoNH spectrum will provide the ppm positions for the carbon resonances (on the Z-axis) and the HSQC TOCSY will provide the ppm positions for the hydrogen resonances (X- and Y-axes). The lines of intersection between the carbon and hydrogen resonances will hopefully show where the peaks (with HHC dimensions) can be found within the HCcH spectrum.

Open window5 to view the HCcH TOSCY spectrum. You can close the other windows to save space. Go to the main Analysis menu and select M: Resonance: Spin Systems. In the Spin Systems table that opens, click on the "Residue" column to sort the spin systems (groups of resonances in a asingle residue) into sequential order. Scroll down to the row for 19Thr, select the row, check that window5 is selected in the top right pulldown menu,  and click [Display Strips]. In window5 you will hopefully see three strips; for the CA-HA, CB-HB & CG2-HG2* positions. Each strip will have a vertical line at its centre, and there will be three horizontal lines, one for each side chain hydrogen. Where the horizontal lines intersect the vertical ones you will see intensity maxima in the HCcH spectrum.

With 19Thr and window5 still selected in the Spin Systems table, click [Display Cells]. You will now see that the display in window 5 is now also split vertically to give separate HG2*, HA, HB and HN regions: these are for all hydrogens in the spin system although the HN resonance is not visible in the HCcH TOCSY, however it would be in a 13C HSQC NOESY. For each of the nine intersection points with obvious maxima pick new peaks ('Shift' + 'Ctrl' + left click). Assign each peak by pressing 'a' with the mouse over the peak cross. The relevant resonances should be automatically presented for you, so all you have to do is  to click on the options in the right hand side in the Assignment: Assignment Panel popup. Note that you can assign several the peaks that align vertically or horizontally to the same resonances (in the matching dimensions) by selecting the peaks (left 'Shift' + left click) and spreading the assignments with R: Assign: Propagate assignments. Note that the 19Thr Ha, Hb, Ca peak at 3.82, 4.26, 66.0 ppm is slightly distorted along the Z-axis, so you may have to manually place the peak in the CA plane before it can be assigned to 19ThrCa; either pick it with 'Ctrl' + left click or select it and press 'p' while in the exact CA plane.

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