Characterization of the simultaneous decay kinetics of metarhodopsin states II and III in rhodopsin by solution-state NMR spectroscopy.

The mammalian visual dim-light photoreceptor rhodopsin is considered a prototype G protein-coupled receptor. Here, we characterize the kinetics of its light-activation process. Milligram quantities of α,ε-(15)N-labeled tryptophan rhodopsin were produced in stably transfected HEK293 cells. Assignment of the chemical shifts of the indole signals was achieved by generating the single-point-tryptophan to phenylalanine mutants, and the kinetics of each of the five tryptophan residues were recorded. We find kinetic partitioning in rhodopsin decay, including three half-lives, that reveal two parallel processes subsequent to rhodopsin activation that are related to the photocycle. The meta II and meta III states emerge in parallel with a relative ratio of about 3:1. Transient formation of the meta III state was confirmed by flash photolysis experiments. From analysis of the site-resolved kinetic data we propose the involvement of the E2 -loop in the formation of the meta III state.

Influence of a joining helix on the BLUF domain of the YcgF photoreceptor from Escherichia coli.

BLUF-domain-comprising photoreceptors sense blue light by utilizing FAD as a chromophore. The ycgF gene product of Escherichia coli is composed of a N-terminal BLUF domain and a C-terminal EAL domain, with the latter postulated to catalyze c-di-GMP hydrolysis. The linkage between these two domains involves a predominantly helical segment. Its role on the function of the YcgF photoreceptor domain was examined by characterizing BLUF domains with and without this segment and reconstituting them with either FAD, FMN or riboflavin. The stability of the light-adapted state of the YcgF BLUF domain depends on the presence of this joining, helical segment and the adenosine diphosphate moiety of FAD. In contrast to other BLUF domains, two-dimensional (1)H,(15)N and one-dimensional (1)H NMR spectra of isotope-labeled YcgF-(1-137) revealed large conformational changes during reversion from the light- to the dark-adapted state. Based on these results the function of the joining helix in YcgF during signal transfer and the role of the BLUF domain in regulating c-di-GMP levels is discussed.

Isotope labeling of mammalian GPCRs in HEK293 cells and characterization of the C-terminus of bovine rhodopsin by high resolution liquid NMR spectroscopy.

High amino acid coverage labeling of the mammalian G protein coupled receptors (GPCR) rhodopsin was established with 15N and 15N/13C isotopes. Rhodopsin was expressed at preparative scale in HEK293S cells and studied in full-length by NMR spectroscopy in detergent micelle solution. This resulted in the assignment and detailed study of the dynamic properties of the C-terminus of rhodopsin. The rhodopsin C-terminus is immobilized until Ala333, after which it becomes unstructured.

Combined solid state and solution NMR studies of alpha,epsilon-15N labeled bovine rhodopsin.

Rhodopsin is the visual pigment of the vertebrate rod photoreceptor cell and is the only member of the G protein coupled receptor family for which a crystal structure is available. Towards the study of dynamics in rhodopsin, we report NMR-spectroscopic investigations of alpha,epsilon-15N-tryptophan labeled rhodopsin in detergent micelles and reconstituted in phospholipids. Using a combination of solid state 13C,15N-REDOR and HETCOR experiments of all possible 13C'(i-1) carbonyl/15N(i)-tryptophan isotope labeled amide pairs, and H/D exchange 1H,15N-HSQC experiments conducted in solution, we assigned chemical shifts to all five rhodopsin tryptophan backbone 15N nuclei and partially to their bound protons. 1H,15N chemical shift assignment was achieved for indole side chains of Trp35(1.30) and Trp175(4.65). 15N chemical shifts were found to be similar when comparing those obtained in the native like reconstituted lipid environment and those obtained in detergent micelles for all tryptophans except Trp175(4.65) at the membrane interface. The results suggest that the integrated solution and solid state NMR approach presented provides highly complementary information in the study of structure and dynamics of large membrane proteins like rhodopsin.

Solution structure and stability of the full-length excisionase from bacteriophage HK022.

Heteronuclear high-resolution NMR spectroscopy was employed to determine the solution structure of the excisionase protein (Xis) from the lambda-like bacteriophage HK022 and to study its sequence-specific DNA interaction. As wild-type Xis was previously characterized as a generally unstable protein, a biologically active HK022 Xis mutant with a single amino acid substitution Cys28-->Ser was used in this work. This substitution has been shown to diminish the irreversibility of Xis denaturation and subsequent degradation, but does not affect the structural or thermodynamic properties of the protein, as evidenced by NMR and differential scanning calorimetry. The solution structure of HK022 Xis forms a compact, highly ordered protein core with two well-defined alpha-helices (residues 5-11 and 18-27) and five beta-strands (residues 2-4, 30-31, 35-36, 41-44 and 48-49). These data correlate well with 1H2O-2H2O exchange experiments and imply a different organization of the HK022 Xis secondary structure elements in comparison with the previously determined structure of the bacteriophage lambda excisionase. Superposition of both Xis structures indicates a better correspondence of the full-length HK022 Xis to the typical 'winged-helix' DNA-binding motif, as found, for example, in the DNA-binding domain of the Mu-phage repressor. Residues 51-72, which were not resolved in the lambda Xis, do not show any regular structure in HK022 Xis and thus appear to be completely disordered in solution. The resonance assignments have shown, however, that an unusual connectivity exists between residues Asn66 and Gly67 owing to asparagine-isoaspartyl isomerization. Such an isomerization has been previously observed and characterized only in eukaryotic proteins.

Persistent bissilylated arenium ions.

A series of bissilylated arenium ions 1 with different substitution patterns on the aryl ring have been synthesized by hydride abstraction from 2-aryl-substituted 2,6-dimethyl-2,6-disilaheptanes (2) via transient silylium ions. The arenium ions have been identified by their characteristic NMR chemical shifts, (delta(29)Si=19.1-25.6, delta(13)C(ipso) =89.0-102.4, delta(13)C(ortho)=160.9-182.0, delta(13)C(meta)=132.5-146.9, delta(13)C(para)=150.2-169.9) supported by quantum mechanical calculations of structures, energies, and magnetic properties at the B3LYP/6-311G(d,p)//B3LYP/6-31G(d) + DeltaZPVE level of theory. The calculations clearly reveal the charge dispersing and stabilizing effect of the silyl substituents in arenium ions 1. The bissilylated benzenium ion 1a is more stable than the parent benzenium ion (C(6)H(7)(+)) by 37.6 kcalmol(-1). The synthesized arenium ions 1 are stable in solution at room temperature for periods ranging from a few hours to days. This unusual stability is attributed to: 1) the thermodynamic stabilization of the arenium ion by two beta-silyl substituents and 2) the essentially non-nucleophilic reaction conditions (the use of the weakly coordinating [B(C(6)F(5))(4)](-) anion and aromatic hydrocarbons as solvents). Addition of stronger nucleophiles than aromatic hydrocarbons (for example, acetonitrile) results in desilylation of the arenium ion 1 and recovery of the 2-aryl-2,6-disilaheptane moiety.