Detecting transient intermediates in macromolecular binding by paramagnetic NMR

Macromolecular complex formation is governed by two opposing constraints of specificity and speed^1,2. Kinetic^3-6 and theoretical considerations suggest that significant rate enhancement can be achieved either by reducing the dimensionality of the search process^1,7 or by the creation of a short-range attractive potential around the target site². This implies the existence of transient intermediates involving non-specific binding modes. Here we show that intermolecular paramagnetic relaxation enhancement (PRE) provides a means of directly detecting the presence of, and investigating the nature of, low population transient intermediates under equilibrium conditions. Applying this approach, we characterize the search process whereby a sequence-specific transcription factor (the homeodomain of HOXD9) binds to non-cognate DNA sites as a means of enhancing the rate of specific association. The PRE data in the fast exchange regime reveal the presence of transient intermediates formed in a stochastic manner at non-cognate sites whose structure is similar to that of the specific complex. Two distinct search processes involving intra- as well as intermolecular translocations can be delineated. The intermolecular PRE method is general and can be readily applied to investigations of transient intermediates in many other macromolecular binding processes.