Amides exchange rates/temperature

Amide proton temperature coefficients and hydrogen exchange rates can provide information about hydrogen-bonding interactions and solvent sequestration in unfolded or partly folded proteins (Dyson and Wright, 1991). Abnormally low temperature coefficients, relative to random coil values, are a clear indication of local structure and interactions. 

Amide hydrogen exchange rates are also dependent on temperature, with approximately three times increase in both acid- and base-catalyzed exchange rates for every increment of 10°C [9] and is described in more detail in Section 1.2.2 (see also Figure 1.6). To minimize variations in hydrogen exchange between multiple experiments, temperature should be precisely controlled ( 2°C)... 

Wagner, G., Wiithrich, K. (1979) Correlation between the amide proton exchange rates and the denaturation temperatures in globular proteins related to the basic pancreatic trypsin inhibitor. 7 Mol Biol, 130 (1), 31-37. 

Labelling with alone can be sufficient to overcome spectral overlap for proteins of up to 20 kDa and, for these proteins, virtually complete resolution can often be achieved for the backbone amide groups in 2D-iH-i HSQC experiments. These experiments are very robust and can be used to determine amide proton exchange rates or chemical shift temperature coefficients. For high protein concentrations H-i HSQC data sets can be acquired rapidly typically within 10 min for a 2 mM sample or 2-3 hr for a 0.2 mM protein sample. Consequently, this experiment has become the mainstay of NMR approaches to monitor the binding of ligands to i -labelled proteins through titration experiments. 

In Eq. (15.3), m is defined as MG / [denaturant], C, denaturant at the SUPREX transition midpoint, R is the gas constant, T is the temperature in kelvin, (Aiint) is the average intrinsic exchange rate of an amide proton, t is the HDX time, n is the number of subunits in the protein, and [P] is the protein concentration expressed in -mer equivalents. The transition region in the SUPREX curve depicts the range of denaturant concentrations where both the native and the unfolded state of protein are significantly populated. 

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