Detecting transient intermediates in macromolecular binding by paramagnetic NMR

Junji Iwahara, G. Marius Clore

Research output: Contribution to journalArticle

294 Scopus citations

Abstract

Macromolecular complex formation is governed by two opposing constraints of specificity and speed1,2. Kinetic3-6 and theoretical considerations suggest that significant rate enhancement can be achieved either by reducing the dimensionality of the search process1,7 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.

Original languageEnglish (US)
Pages (from-to)1227-1230
Number of pages4
JournalNature
Volume440
Issue number7088
DOIs
StatePublished - Apr 27 2006
Externally publishedYes

ASJC Scopus subject areas

  • General

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