Direct Millisecond Hydrogen Exchange

The nmr data for this type of motion are direct and the motion clearly involves rotation about bonds in the millisecond time scale range. However. less direct evidence for motion comes from other techniques such as fluorescence depolarization, 02 diffusion, hydrogen exchange kinetics, and nmr relaxation times (see Ref. 4). The extent of this motion is not yet easy to define, but this evidence points to motion in the nanosecond time scale range. It is tempting to see the motion in this time scale as bond oscillations rather than rotations. To put it in a different way, on this time scale the side chains have some freedom to move with respect to each other but not normally to undergo substantial bond rotation. Table IV summarizes some references for motion of different types. Additionally, nmr relaxation studies suggest that the backbone or main chain of a protein is more restricted than that of the side chains. 

In a standard hydrogen exchange experiment (see Section 2.3.4 for variations in experimental workflow), the sample is deuterated for a predetermined time (milliseconds and longer times), and the reaction is slowed down several orders of magnitude by the addition of an acidic quench solution. Quenched reactions can be subjected to direct mass spectrometry analysis or immediately flash frozen in liquid nitrogen and stored at -80°C in multiple aliquots for future analysis. These flash-frozen samples need to be thawed prior to mass spectrometry. It is important to note that freeze-thaw cycles contribute to signal loss due to back-exchange (this is described further in Section 2.3.8), which... 

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