What pulse sequences are implemented in TITAN?
The following pulse sequences are implemented in TITAN: HSQC, HMQC, HZQC/HDQC, gHMQC, CPMG-HSQC, CPMG-gHMQC, COSY, and NOESY pulse sequences.
However, to the extent that relaxation or chemical exchange during magnetization transfer steps can be neglected, other experiments such as transverse relaxation-optimized spectroscopy (TROSY) and sensitivity-enhanced HSQCs may still be analyzed as if acquired with a regular HSQC pulse sequence, but the user should be aware of the additional assumptions involved in such an analysis.
What is the optimal shape and selection of a ROI?
ROIs should extend approximately two to three linewidths from the center of resonances and contain the entire region of the spectrum within which resonances are observed across the titration.
Overly large ROIs will result in increased noise, slower fitting calculations, and potentially inadvertant overlaps with adjacent resonances. Conversely, tightly cropped ROIs will reduce the accuracy of fitted linewidths.
What should the concentrations of my samples be?
Optimal results are obtained when the protein concentration is comparable to Kd. Protein concentrations of at least 10 μM are required, but higher protein concentrations, around 50-100μ M, will generally allow sufficiently high signal-to-noise ratios to be obtained in less than an hour. The final ligand concentration should be sufficient to fully saturate the binding: the greater of at least three times the Kd or two equivalents relative to the protein concentration.
How many titration points should be acquired?
Approximately 8-12 titration points should be acquired. However, these guidelines are based on a simple 1:1 association reaction, P + L ⇌ PL. The identification and analysis of more complex binding mechanisms may benefit from a greater number of points or, for example, varying the protein concentration in addition to that of the ligand.