Sequential protein backbone assignments

Introduction

We will now look at the sequentially assigning protein backbone spin systems using triple-resonance experiments. There are two basic, although linked, parts to the process. The first is the linking of sequential spin systems (collections of resonances that relate to one residue) on the basis of matching peak positions. The second is the matching of runs of unassigned spin systems to residues within a sequence.

Spectrum setup

First make sure that window2 is visible and expanded along one side of the screen. With the mouse over window2, select R: Window: Clone. This will produce an identical window containing the triple resonance backbone spectra. Now arrange window2 and the new window5 so that they are side by side, with window2 shrunk to about one quarter the width of window5 (both windows should be as tall as possible) (see figure below). Then switch off the HNCA spectrum in window2, via the "Spectrum" tab, so that only the HNcoCA is visible.

Now Select M: Assignment: Protein Sequence Assignment. You should start out in the {Windows & Spectra} tab. Select window2 as the Query "13C window:" and window5 as the Match "13C window:". Set |Use| to True for "HNcoCA:111" in the top Query panel and for "HNCA:110" in the "Matches" panel (see figure below). This setup means that we are going to compare specified 13C peak positions in the HNcoCA experiment with potentially matching peak positions in the HNCA experiment.

The rationale here is that the HNcoCA experiment's peaks have the carbon shift of the preceding alpha carbon along the polypeptide chain at a given amide location, where the HNCA has both the intra-residue and preceding alpha carbon peaks for each amide. Thus we can potentially use both spectra to say two amide spin systems are sequentially connected by saying that an inter-residue peak of the HNcoCA derives from the same resonance as an intra-residue peak of the HNCA.

Note that this system can readily use other backbone experiments like HNcoCACB, HNCO, HB/HAcoNH etc. with the same approach and that this tutorial only uses the alpha carbon experiments for simplicity. Follow the Protein assignment tutorial to learn more about this.

Linking sequential spin systems

Now go to the {Spin System Table} tab and click on the row that corresponds to spin system {90}. You will see that the two triple resonance windows move to new locations. The location of window2 is at spin system {90} as found in the HNcoCA. The window with the HNCA, window5, has moved to the position of any peaks that match the alpha carbon frequency. In this case there is only one good match, which corresponds to spin system {89}:

Note that if the "Filter by Inter/Intra Type" option in the {Options} tab is set to off then spin system {90} matches two HNCA peaks, but we know that the spin system {89} peak is the correct one because the 76Lys peak is already assigned and because the 76Lys HNCA peak is not an intra-residue one, i.e. it coincides with the 76Lys HNcoCA peak. Click to highlight the row corresponding to spin system {89} in the Match Peak panel, and then click [Set Seq Link]. You will now see that the tables of the popup update to show that {90} is set as "i+1" of {89}:

Also note that in the spectrum window the peak annotations of the aligned  HNcoCA and HNCA peaks have changed to illustrate that they are both assigned to the same 13C resonance. Now click [Goto i-1], which will repeat the carbon shift matching, but this time from a sequence position one earlier in the sequence. For spin system {89} apply [Set Seq Link] for the only match to {171}, then [Goto i-1] again. Repeat the sequential linking procedure for {171} & {161}:

Assigning segments to the sequence

You will see that as you select the various spin systems in the main table, in the lower right 'Residue Types' table there is a display of the probable types of amino acid residues. This prediction is based upon how well the shifts within the spin system match the chemical shifts in the BMRB database. As spin systems are connected sequentially, amino acid type predictions are made for the whole sequentially connected section. In the lower left hand table, 'Sequence Locations', the connected spin systems, given their probably amino acid types, are matched to the protein sequence. Here the highest scoring positions of residue type match for various five residue sections are listed. You will see that the assigned residue positions are coloured blue, but that there is a grey section (starting at 8Ser) which is not assigned that matches the section we have just linked with high probability. Simply select the row for this unassigned selection and click [Assign Selected]. You will see that all the residues in the section (by virtue of their links for the most part) become assigned to the selected section:

Select the option M: Molecule: Atom Browser. Make sure that the element [H] is displayed (click the button to get the green hydrogen assignment options) and look at the "H" (amide) atom for 8Ser. You will see that not only is 8Ser assigned (i.e. the atom option goes dark green), but the atoms in the residues which we just connected sequentially are also assigned:

Go through the spectra using the Assignment: Protein Sequence Assignment popup with [Goto i+1] to verify that all the connected spin systems have been assigned. Note that in this instance it was possible to assign the resonances to unique atoms, as well as assign the backbone spin systems to the sequence, because the resonances had their atom type set previously.

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