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Styrene-maleic acid (SMA) copolymers solubilize biological membranes to form lipid nanoparticles (SMALPs) that contain membrane proteins surrounded by native lipids, thus enabling the use of a variety of biophysical techniques for structural and functional studies. The question of whether SMALPs provide a truly natural environment or SMA solubilization affects the functional properties of membrane proteins, however, remains open. We address this question by comparing the photoactivation kinetics of rhodopsin, a G-protein-coupled receptor in the disk membranes of rod cells, in native membrane and SMALPs prepared at different molar ratios between SMA(3:1) and rhodopsin. Time-resolved absorption spectroscopy combined with complex kinetic analysis reveals kinetic and mechanistic differences between the native membrane and SMA-stabilized environment. The results suggest a range of molar ratios for nanoparticles suitable for kinetic studies.

Membrane proteins often require solubilization to study their structure or define the mechanisms underlying their function. In this study, the functional properties of the membrane protein rhodopsin in its native lipid environment were investigated after being solubilized with styrene-maleic acid (SMA) copolymer. The static absorption spectra of rhodopsin before and after the addition of SMA were recorded at room temperature to quantify the amount of membrane protein solubilized. The samples were then photobleached to analyze the functionality of rhodopsin upon solubilization. Samples with low or high SMA/rhodopsin ratios were compared to find a threshold in which the maximal amount of active rhodopsin was solubilized from membrane suspensions. Interestingly, whereas the highest SMA/rhodopsin ratios yielded the most solubilized rhodopsin, the rhodopsin produced under these conditions could not reach the active (Meta II) state upon photoactivation. The results confirm that SMA is a useful tool for membrane protein research, but SMA added in excess can interfere with the dynamics of protein activation.

All but two out of 13 cysteine residues in CB1 (C257 and C264) can be replaced with alaniens and still yield a functionally active reporter. It is demonstrated that the substituted cysteine accessibility method can be carried out to study the structure and function of the CB1 receptor.

Research has been conducted on the proteins. The ability of the proximal tryptophan residues to cause bimane quenching in proteins has been investigated and the results indicate that this property can be used in mapping the distances between tryptophan and bimane probes at the protein strategic sites.

Research describes mutations in the retinal binding pocket of rhodopsin that will render the protein to retain its normal functional properties under ambient lighting conditions. Results demonstrate a triple mutant that exhibits a large blue-wavelength shift with an absorbance maxima at wavelength 453.

Site-directed mutagenesis can be used to introduce a fluorescent probe into a protein to study its structure and function. Researchers describe a technique using cysteine substitutions labeled with the fluorescent probe monobromobimane.

The localization of cysteines 65 and 316 in the dim light photoreceptor rhodopsin was investigated in cysteine mutant bovine rhodopsin expressed in COS-1 cells. After derivatizing the cysteines with N-alkylmaleimides and, spin labels, electron paramagnetic resonance spectroscopy revealed that in the dark state cysteine 65 and 316 were in proximal locations but moved to opposite directions upon photoexcitation.

Previous studies on systematic single cysteine substitutions of the amino acids in the cytoplasmic C-D loop were extended by replacing, one at a time, the amino acids Q225-I256 in the E-F loop region by cysteines. All the mutants formed the characteristic rhodopsin chromophore with 11-cis-retinal. While most of the mutants bleached normally, L226C, indicated abnormal bleaching behavior. Some mutants, especially L226C, had low reactivity. Findings indicated a specific structure in the E-F loop that interacts with transducin.

Thirty consecutive single cysteine substitution mutants in the amino acids Q225-I256 of bovine rhodopsin were prepared and modified with sulfhydryl specific nitroxide reagent. This sequence includes the E-F interhelical loop, a transducin interaction site. The accessibilities of the attached nitroxides to collisions with hydrophilic and hydrophobic paramagnetic probes in solution were determined. Findings indicated that chromophore isomerization results in patterns of structural modifications that can be interpreted in terms of movements of helices that extend into the aqueous loop regions.

Time-resolved absorption measurements are performed on the forward phototransformation of native oat phytochrome at 10 degrees C. Likewise, laser photolysis allows for the measurement of the transient difference absorption spectra at delay times from 100 ns to 800 ms. A global fitting algorithm, together with a singular value decomposition method, is used to evaluate the absorption changes at different wavelengths which follow the same kinetics.

The time-resolved circular dichroism (TRCD) spectra of the intermediates formed during the Pr -> Pfr phototransformation in native oat phytochrome is measured at 10 degrees C. The spectra shows the presence of the lumi-R intermediate whose TRCD spectrum is substantially different from its steady-state CD spectrum at low temperature. This discrepancy may be due to different protein-chromophore interactions. It is proposed that the lumi-R trapped in the low-temperature experiment is an unrelaxed isomerized species.