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The classical view of DNA posits that DNA must be stiff below the persistence length (<150 bp) but recent studies addressing this have yielded contradictory results. We developed a fluorescence-based, protein-free assay for studying the cyclization of single DNA molecules in real time. The assay samples the equilibrium population of a sharply bent, transient species which is entirely suppressed in single molecule mechanical measurements and is biologically more relevant than the ligated species sampled in the traditional ligase-based assay. The looping rate has a remarkably weak length dependence between 67 and 106 bp that can not be described by the worm-like chain model. Many biologically significant protein-DNA interactions that involve looping and bending of DNA below 100 bp likely use this intrinsic bendability of DNA.

Small multidrug resistance (SMR) transporters provide an ideal system to study the minimal requirements for active transport. EmrE is an E. coli SMR transporter that exports a broad class of polyaromatic cation substrates, thus conferring resistance to drug compounds matching this chemical description. However, a great deal of controversy has surrounded the topology of the EmrE homodimer. Here we show that asymmetric antiparallel EmrE exchanges between inward- and outward-facing states that are identical except that they have opposite orientation in the membrane. We quantitatively measure the global conformational exchange between these two states for substrate-bound EmrE in bicelles using solution NMR dynamics experiments. FRET reveals that the monomers within each dimer are antiparallel, and paramagnetic relaxation enhancement NMR experiments demonstrate differential water accessibility of the two monomers within each dimer. Our experiments reveal a “dynamic symmetry” that reconciles the asymmetric EmrE structure with the functional symmetry of residues in the active site.

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors in eukaryotes. Crystal structures have provided insight into GPCR interaction with ligands and G-proteins1,2, but our understanding of the conformational dynamics of activation is incomplete. Metabotropic glutamate receptors (mGluRs) are dimeric class C GPCRs that modulate neuronal excitability, synaptic plasticity, and serve as drug targets for neurological disorders3,4. A “clamshell” ligand-binding domain (LBD), which contains the ligand binding site, is coupled to the transmembrane domain (TMD) via a cysteine rich domain, and LBD closure appears to be the first step in activation5,6. Crystal structures of isolated mGluR LBD dimers led to the suggestion that activation also involves a reorientation of the dimer interface from a “relaxed” to an “active” state7,8, but the relationship between ligand binding, LBD closure and dimer interface rearrangement in activation remains unclear. We used single-molecule fluorescence resonance energy transfer (smFRET) to probe the activation mechanism of full-length mammalian group II mGluRs. We find that the LBDs interconvert between three conformations: resting, activated and a short-lived intermediate state. Orthosteric agonists induce transitions between these conformational states with efficacy determined by occupancy of the active conformation. Unlike mGluR2, mGluR3 displays basal dynamics, which are Ca2+ dependent and lead to basal protein activation. Our results support a general mechanism for the activation of mGluRs in which agonist binding induces closure of the LBDs followed by dimer interface reorientation. Our experimental strategy should be widely applicable to study conformational dynamics in GPCRs and other membrane proteins.