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NOTE FOR INSTRUCTORS (Georgia State and Georgia Tech):

Nothing new to install for this week.

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GOALS:

1) More on sequential assignment principles using 3D HNCO, 3D HNCACB and 3D CBCACONNH experiments.
 
 

INTRODUCTION TO NMRView:
 

    The NMRView is a software developed by Bruce A. Johnson at Merck Research Laboratories for vizualisation and analysis of multidimensional NMR data.  For this class we will learn the basics of NMRView and next week, we will use it for sequential assignment of a 24-amino acid peptide using triple resonance experiments.
 
 

THE N-PEPTIDE:

We will work on the sequential assignment of the N-peptide in complex with an RNA. For more info on this RNA-peptide complex:  Legault et al. Cell 1998, vol.93, 289-299.
 

The sequence of the N-peptide is:

-2 -1   1     2    3   4    5   6    7   8    9   10 11  12 13  14  15  16  17 18   19  20  21  22
 g   s   M   D   A   Q   T   R   R   R   E   R   R   A   E   K   Q   A   Q   W   K   A   A   N
 
 

3D DATASETS FOR PROTEIN SEQUENTIAL ASSIGNMENTS
 

1. Start NMRView by typing:

%cd  /21/users/pascale/bcmb8190_00/nmrview
%nmrview -- -b MYDatabase5.str

2.  In the Console window type:

>source hsqc.tcl
>seqass
>source jump.tcl
 

You will get 4 windows, each one containing a single dataset: The same hsqc experiment, and hnco, a hncacb, and a cbcaconnh. I have already created a peak list for each of these experiments.

3.  Expand the hsqc window around peak 14 and place the black cursor in the middle of peak 14. Type j (for jump) on your keyboard. This simple command selects the proper ¹⁵N plane in the three 3D spectra (117.8 ppm) and positions the black cursor at the same proton frequency as the HSQC crosspeak. Note that the cursors in the various windows track each other automatically.

For sequential assignment purposes, the hsqc gives the ¹H and ¹⁵N frequencies of residue i, the hnco gives the C' frequency of residue i-1, the cbcaconnh gives the Ca and Cb of residue i-1, and the hncacb gives the Ca and Cb frequencies of residues i-1 and i.

We can tell from the Cb frequency that residue i-1 is an alanine. It could be Ala3, Ala12, Ala16, Ala20, or Ala21. The Cb chemical shift at 28.5 ppm indicate that residue i is either a CYS (not present in N-peptide), a GLU, a HIS (not present in N- peptide), a GLN, an ARG, or a TRP. That means that the only possibilities for these two residues are Ala3-Gln4, Ala12-Arg13, or Ala16-Gln17.

For a table of ¹³C chemical shift see Cavanagh et al. Protein NMR Spectroscopy" p. 544.  Note that there was a mistake on that page in the original version of the book. To get corrections, go to Art Palmer web's site: http://cpmcnet.columbia.edu/dept/gsas/biochem/labs/palmer/protein_nmr_spectroscopy/.  For this lab you can more simply access the following link:¹³C chemical shifts.

4.  Going to the next residue is a little more tricky, but not very difficult with a small peptide like this.

Note the Ca and Cb chemical shifts of residue i from the hncacb spectrum.

Ca:   57.5 ppm
Cb:   28.5 ppm

In the hncacb window, position the black cursor in the center of Ca and type "n" (for next) on the keyboard. This command will redisplay the cbcaconnh experiment at the carbon frequency 57.5 ppm. The red boxes indicate the peaks that have maximum intensity on this carbon plane. To go up and down the planes, use the arrow keys. Note the frequencies of the crosspeaks at this carbon plane and at the carbon planes above and below (this is to allow for errors in ¹³C chemical shift).

at 57.5 ppm:  8.38 118.8
                    7.70 121.5
                    7.54 120.6

at 57.05 ppm:   none

at 57.95 ppm:   8.15 122.5

Back to the hncacb window, position the black cursor in the center of Cb and type "n" on the keyboard. This will redisplay the cbcaconnh experiment at the carbon frequency 28.5 ppm. Record the frequencies of the crosspeaks at this carbon plane and at the carbon planes above and below.

at  28.5 ppm:  7.58 119.5
                     7.99 120.1

at  28.95 ppm:  8.42 119.9
                       8.01 120.7

at  28.05 ppm:  7.70 121.5

The peak at 7.70 121.5 is observed for both ¹³C planes (Ca and Cb) and in this case unambiguously identifies the amide proton and nitrogen of the next residue in the sequence.

5.  Back to the hsqc, expand the window around peak #20 (7.70 121.5 ppm). Place the black cursor in the center of that peak and type "j" on the keyboard. See above, for the information obtained in each spectrum. Importantly, the hncacb gives the Ca and Cb chemical shifts of the next residue in the sequence (i+1) at 60.6 ppm and ~28.7 ppm. the Cb chemical shift indicate that this residue is either a Glu, Gln, Arg, or Trp. You get the idea and can continue these assignments on your own....

Please note that I design this simple strategy to obtain sequential assignments on my own. Sequentail assignments can be done in a more automated way, but the principle would be the same. Look at CBCA analysis in the NMRView Users Manual for another method for sequential assignment or write your own!

6.  Complete the peak and assignment tables.

We have already seen how to manually fill the peak tables.

An empty assignment table can be created by entering a sequence file, a psf file, or a pdb file of your molecule. The assignment table can be seen under "Analysis", "Assignments". It can be filled manually or via some tcl-defined processes.
 

LAB PROBLEM SET #10:
 

1.  What are the three sequential residues of the N-peptide that were assigned during the lab today?