Centenary Award and Sir Frederick Gowland Hopkins Memorial Lecture 92-0520, sandis μ 2013 Biochemical Society 1343 -peptide with the C β The Authors Journal compilation C There are three NMR techniques that can be used dependent on distances between a paramagnetic label and Introduction Proteins and their complexes areare not best static described but as dynamic an ensemble and is of states. located The at major species the minimumrepresents of the the species free that energysuccess has landscape by and been studied conventionalniques, with structural including so crystallography and much andpicture biophysical NMR. But revealed tech- the static bycomplete picture. these In addition to studies the majorhighly species, transient does sparsely there populated exist not states that ariseexcursions describe from between rare the the minimum freeand other energy local configuration minima of the free energythat landscape [1]. the Given populations of suchlow sparsely populated that states they are so arewhy effectively they invisible, should one evenbiological might be processes, well studied. including ask Itbinding, molecular turns allostery, recognition out that and conformationaland self-assembly, many proceed via selection, such sparsely populated states. But induced little is known fit about the structuralpopulated properties states, of since sparsely they are difficultinaccessible to trap (i.e. and invisible) therefore toadvances conventional methods. in Recent solution NMRdoor to directly spectroscopy studying sparsely populated have states of opened proteins and their the complexes. to investigate sparselyranging populated from 0.5 states to 5% withto and milliseconds. lifetimes PRE occupancies spanning (paramagnetic relaxation nanoseconds enhancement) probes states with lifetimes less than 250–500 scale interdomain motionsto involved the in interaction of ligandsurface monomeric of binding, amyloid amyloid protofibrils and and the internalthe cavity chaperonin surface GroEL. of Centenary Award and Sir Frederick GowlandLecture HopkinsDelivered Memorial at the MRC LaboratoryMolecular of Biology, Cambridge,13 December on 2012 Marius Clore -peptide; CaM, calmodulin; DEST, dark-state exchange β 1 ,amyloid β A
conformational selection, encounter complex, induced fit, macromolecular assembly,
email [email protected] Biochem. Soc. Trans. (2013) 41, 1343–1354; doi:10.1042/BST20130232 protein–DNA interaction, sparsely populated state. Abbreviations used: 1 Key words: Sparsely populated transientcomplexes play an states important role of inincluding many biological proteins protein–protein processes and andlostery, conformational selection, their induced protein–DNA fit and self-assembly. recognition,These al- states areand difficult transient to nature studyconventional as makes structural their them andpresent low article, biophysical effectively I population summarize techniques. invisible recentlaboratory, NMR In including to developments the in the use our hancement, of lifetime line paramagnetic broadening relaxation andsaturation en- dark-state exchange transfer spectroscopy,sparsely that populated states have toin permitted be some such detected, instances, characterizedthese visualized. and, methods I illustrate totranscription the the factors locate application elucidation their of specific ofoverwhelming target sea mechanisms sites of within non-specific whereby DNA, an of to encounter the complexes in characterization protein–protein recognition, to large- Abstract
*Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 208 diamagnetic NMR G. Marius Clore* Seeing the invisible by paramagnetic and U.S.A. Gowland Hopkins Memorial Lecture Centenary Award and Sir Frederick saturation transfer; EIN,protein; N-terminal MBP, domain maltose-bindingphosphotransferase system; protein; of Rubisco, ribulose-1,5-bisphosphate PRE, carboxylase/oxygenase. Enzyme paramagnetic I; relaxation HPr, enhancement; histidine PTS, phosphocarrier Biochemical Society Transactions www.biochemsoctrans.org Transactions Society Biochemical 1344 Biochemical Society Transactions (2013) Volume 41, part 6
protons being shorter in the minor species than the major In a system in which there is rapid exchange between one [2,3]. Relaxation dispersion spectroscopy probes states various species, the large magnitude of the PRE at short with lifetimes typically ranging from 0.5 to 10 ms (although distances can be taken advantage of to detect sparsely in particularly fortunate cases, lifetimes as short as 50 μs can populated states [2,3]. For example, let us say that we have be reached) and is dependent on significant chemical shift a system comprising a major species with an occupancy differences between the resonances of the major and minor of 99% and a paramagnetic centre–proton distance of 30 species [4–6]. Lastly, DEST (dark-state exchange saturation A˚ , and a minor species at an occupancy of 1% with a transfer) spectroscopy and lifetime line broadening can probe corresponding distance to the same proton of 8 A˚ .For events on timescales ranging from 500 μs to 100 ms and are a ∼20 kDa system, the PRE rate will have a value of ∼2s− 1 dependent on very large differences in transverse relaxation for the major species and ∼5600 s − 1 for the minor species. obs (R2) rates (i.e. linewidths) between the major and minor The dependence of the observed PRE rate, 2 , measured species [7,8]. on the spectrum of the major species, can be calculated In the present article, I first describe the underlying basis exactly using the McConnell [14] equations. In the fast
for the use of the PRE to detect sparsely populated states exchange regime, where the exchange rate kex between the two and illustrate its application to protein–DNA and protein– species is significantly (>10-fold) larger than the difference obs ∼ − 1 protein interactions, as well as to the investigation of the in 2 values, 2 ( 51 s ) will simply be given by the interplay between conformational selection and induced fit. population weighted average of the 2 rates for the major and I then describe the basis for lifetime line broadening and minor species. Under these conditions, the footprint of the DEST to study interactions between NMR visible molecules spectroscopically invisible minor species will be manifested and large macromolecular assemblies, and illustrate this by on the PRE profiles measured on the NMR resonances of studies on the interaction of soluble monomeric Aβ (amyloid the major species, providing there are paramagnetic centre– β-peptide) with amyloid protofibrils and the prototypical proton distances that are shorter in the minor species than chaperonin GroEL. the major one. Even if exchange between the species is in the intermediate regime where the difference in
2 values is approximately equal to kex, the fast exchange obs approximation can still be used even though 2 may be a Fundamentals of the PRE factor of two smaller than the population weighted average. The PRE is caused by magnetic dipolar interactions between This is due to the
rate, 2, by taking the difference in transverse relaxation rates specific site centrally located [2] (Figure 1). The equilibrium (R2) between the paramagnetic sample and a diamagnetic dissociation constant at 100 mM NaCl is 1.5 nM with 2 + 2 + − 1 control (e.g. Mn compared with Ca ) [12,13]. Because the a dissociation rate constant koff of less than 0.01 s . paramagnetic centre is generally attached to macromolecules Intermolecular PRE data for 15N-labelled HoxD9 were via a flexible linker, the original PRE formalism of Solomon recorded using an EDTA–Mn2 + paramagnetic tag located [9] and Bloembergen and Morgan [10] has to be extended to at four different sites (individually) on the DNA (Figure 1A). a model-free formalism to take into account the influence of At low salt, the PRE profiles from the four sites are in internal motions of the paramagnetic label on the PRE [12]. excellent agreement with the structure of the specific complex The availability of long-range distance information from PRE (Figures 1C and 1E). As the salt is raised to 100 or 160 mM measurements can obviously be very useful in NMR structure NaCl, the PRE profiles bear no resemblance to those expected determination, especially as one can refine directly against the for the specific complex (Figures 1D and 1F); furthermore, the PRE data [12]. PRE profiles for sites 1 and 4 (paramagnetic labels located at