Following chemical shifts (1)

Introduction

Firstly we will look at the situation where you have a series of spectra which are related to one another by virtue of the fact that some experimental condition is changing, which is causing the peak positions to move in response to that changing condition.

Typically this would be a titration where there is an increase in concentration of a ligand that one hopes binds to the protein sample, a pH series or a temperature series. In this instance for simplicity we will be looking at a temperature series, and aim to measure the temperature coefficient of some of the amide resonances. For other types of series the procedure within the program is broadly the same except that the relation between peak position and chemical shift follows a different relationship; i.e. we fit different types of curve/function to the data.

Defining the NMR series

Open the TutorialProject2 project. To start with have a look at window1, and you will see four spectra, coloured blue, green, orange and red. These represent 15N-HSQC spectra recorded at 288, 298, 303 and 310 K respectively. Note that only one of these spectra is assigned. (And for brevity we have only assigned a few peaks):

Before we begin the chemical shift analysis, we must group these spectra in to what we refer to as an Experiment Series. This means to let Analysis know which spectra are grouped together and how the condition parameter varies across the range. Accordingly go to M: Experiment: NMR Series. In the resultant popup window you will see that we already have one experiment series setup, which is a T1 relaxation series. We will come to this series later on. For now click [Add Series], double click the |Condition Type| column for the newly added row and set the value to "temperature" (at the bottom). This sets up a container for experiments so that we know what value varies, now we specify how the value varies.

Next, click [Add Condition Point] FOUR TIMES, then in the lower 'Sample Conditions' table, for all the newly added rows, double-click in the |Experiment| column, and set the four experiments to H[N]-310K, H[N]-303K, H[N]-298K and H[N]-288K. The order of experiment select is unimportant. Next, double click the |Value| column to formally enter the temperature at which the experiment was recorded. And as you may have already guessed we have cunningly given a hint to which temperature goes with which experiment in its name. Thus enter the Values 310, 303, 298, 288 for the appropriate rows:

In this instance we will neglect filling in the |Error| column, but if you have some measure of precision in the |Value| you could enter it here and Analysis would estimate the error in the temperature coefficient. Now close the Experiment: NMR Series popup.

Briefly look in the M: Experiment: Experiments popup, and look at the {Experiments} tab. You should note that each experiment in our temperature series is associated with a different chemical Shift List. This is because we positively expect the chemical shifts of any resonance (i.e. group of atoms) to differ under the different conditions. It would be inappropriate for the chemical shift values to be averaged over all these experiments, which is what would happen if they only use one Shift List (i.e. shift table). If you forget to set the different Shift Lists for the experiments in a series, rest assured that Analysis will warn you, and give the option to allocate the individual lists, rather than contaminate the existing shift lists.

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