Termination of G protein-coupled receptor (GPCR) signaling typically involves phosphorylation of the receptor, followed by binding of a protein called arrestin. Here we tested the minimal stoichiometry required for this interaction, by determining if a single rhodopsin molecule can bind arrestin. To do this, we prepared nanoscale phospholipids particles, so-called nanodiscs, which contain only monomeric rhodopsin and measured their ability to bind visual arrestin. Our data clearly show that visual arrestin can bind to monomeric phosphorylated rhodopsin to stabilize its active form, called metarhodopsin II. Interestingly, we find beta-arrestin can also bind to monomeric rhodopsin in nanodiscs and stabilize metarhodopsin II. Together, these results suggest that in general, the minimal unit for arrestin binding is a monomeric GPCR.

We have tested whether arrestin binding requires the G-protein-coupled receptor be a dimer or a multimer. To do this, we encapsulated single-rhodopsin molecules into nanoscale phospholipid particles (so-called nanodiscs) and measured their ability to bind arrestin. Our data clearly show that both visual arrestin and β-arrestin 1 can bind to monomeric rhodopsin and stabilize the active metarhodopsin II form. Interestingly, we find that the monomeric rhodopsin in nanodiscs has a higher affinity for wild-type arrestin binding than does oligomeric rhodopsin in liposomes or nanodiscs, as assessed by stabilization of metarhodopsin II. Together, these results establish that rhodopsin self-association is not required to enable arrestin binding.

Studying the interplay between protein structure and function remains a daunting task. Especially lacking are methods for measuring structural changes in real time. Here we report our most recent improvements to a method that can be used to address such challenges. This method, which we now call tryptophan-induced quenching (TrIQ), provides a straightforward, sensitive, and inexpensive way to address questions of conformational dynamics and short-range protein interactions. Importantly, TrIQ only occurs over relatively short distances (∼5-15 Å), making it complementary to traditional fluorescence resonance energy transfer (FRET) methods that occur over distances too large for precise studies of protein structure. As implied in the name, TrIQ measures the efficient quenching induced in some fluorophores by tryptophan (Trp). We present here our analysis of the TrIQ effect for five different fluorophores that span a range of sizes and spectral properties. Each probe was attached to four different cysteine residues on T4 lysozyme, and the extent of TrIQ caused by a nearby Trp was measured. Our results show that, at least for smaller probes, the extent of TrIQ is distance dependent. Moreover, we also demonstrate how TrIQ data can be analyzed to determine the fraction of fluorophores involved in a static, nonfluorescent complex with Trp. Based on this analysis, our study shows that each fluorophore has a different TrIQ profile, or "sphere of quenching", which correlates with its size, rotational flexibility, and the length of attachment linker. This TrIQ-based "sphere of quenching" is unique to every Trp-probe pair and reflects the distance within which one can expect to see the TrIQ effect. Thus,TrIQ provides a straightforward, readily accessible approach for mapping distances within proteins and monitoring conformational changes using fluorescence spectroscopy.

We previously showed that the dramatic quenching of bimane fluorescence by Trp can be exploited to study protein structure, dynamics and protein-protein interactions. Here we report our efforts to extend this approach to four other probes: monobromotrimethylammoniobimane (qBBr), lucifer yellow (LY), BODIPY 507/535 (BDPY), and Atto-655 (Atto). We attached each probe to a unique reactive cysteine at four different sites on T4 lysozyme, and measured the effect of a nearby Trp on the fluorescence intensity and lifetime. We also used a relatively novel yet simple approach to calculate the relative fraction of each probe forming a static, non-fluorescent complex with the Trp. The different probes show markedly different sensitivities to quenching by the Trp, most likely due to their differences in size, rotational flexibility, length of attachment linker, and efficiencies of quenching photochemistry. Together, our data can be used to approximate the distance-dependent “sphere of quenching” for each probe. The smaller probes (bimanes and BODIPY) show substantial quenching and static-complex formation only when they are very close to the Trp (< 10Å away). Thus, these probes are best suited for measuring short-range intramolecular distances or conformational changes in proteins. In contrast, the larger probes (lucifer yellow and Atto-655) show substantial quenching and static-complex formation with the Trp over longer distances. Thus, these latter probes are less selective for monitoring local changes, but are useful for monitoring longer-range distances within a protein, or inter-molecular distances between proteins. We anticipate the broad excitation and emission range covered by these four probes (380 nm - 650 nm), combined with the ability to determine the amount of static complex formation with Trp, should expand the use of Trp-induced fluorescence quenching as a method for studying protein structure and dynamics.

The manganese transport regulator (MntR) from Bacillus subtilis binds cognate DNA sequences in response to elevated manganese concentrations. MntR functions as a homodimer that binds two manganese ions per subunit. Metal binding takes place at the interface of the two domains that comprise each MntR subunit: an N-terminal DNA-binding domain and a C-terminal dimerization domain. In order to elucidate the link between metal binding and activation, a crystallographic study of MntR in its metal-free state has been undertaken. Here we describe the structures of the native protein and a selenomethionine-containing variant, solved to 2.8 A. The two structures contain five crystallographically unique subunits of MntR, providing diverse views of the metal-free protein. In apo-MntR, as in the manganese complex, the dimer is formed by dyad-related C-terminal domains that provide a conserved structural core. Similarly, each DNA-binding domain largely retains the folded conformation found in metal bound forms of MntR. However, compared to metal-activated MntR, the DNA-binding domains move substantially with respect to the dimer interface in apo-MntR. Overlays of multiple apo-MntR structures indicate that there is a greater range of positioning allowed between N and C-terminal domains in the metal-free state and that the DNA-binding domains of the dimer are farther apart than in the activated complex. To further investigate the conformation of the DNA-binding domain of apo-MntR, a site-directed spin labeling experiment was performed on a mutant of MntR containing cysteine at residue 6. Consistent with the crystallographic results, EPR spectra of the spin-labeled mutant indicate that tertiary structure is conserved in the presence or absence of bound metals, though slightly greater flexibility is present in inactive forms of MntR.