RESUMEN
Integral membrane proteins pose considerable challenges to mass spectrometry (MS) owing to the complexity and diversity of the components in their native environment. Here, we use native MS to study the post-translational maturation of bacteriorhodopsin (bR) and archaerhodopsin-3 (AR3), using both octyl-glucoside detergent micelles and lipid-based nanoparticles. A lower collision energy was required to obtain well-resolved spectra for proteins in styrene-maleic acid copolymer (SMA) Lipodisqs than in membrane scaffold protein (MSP) Nanodiscs. By comparing spectra of membrane proteins prepared using the different membrane mimetics, we found that SMA may favor selective solubilization of correctly folded proteins and better preserve native lipid interactions than other membrane mimetics. Our spectra reveal the correlation between the post-translation modifications (PTMs), lipid-interactions, and protein-folding states of bR, providing insights into the process of maturation of the photoreceptor proteins.
Asunto(s)
Proteínas de la Membrana , Nanopartículas , Membrana Dobles de Lípidos , Lípidos , Espectrometría de Masas , MicelasRESUMEN
We demonstrate that (13)C-detected spectra recorded using fast (60 kHz) magic angle spinning on sub-milligram (<10 µmol) quantities of a protonated 7 trans-membrane helix protein (bacteriorhodopsin) in its native lipid environment are comparable in sensitivity and resolution to those recorded using 15-fold larger sample volumes with conventional solid state NMR methodology. We demonstrate the utility of proton-detected measurements which yield narrow (1)H linewidths under these conditions, and that no structural alterations are observed. We propose that these methods will prove useful to gain structural information on membrane proteins with poor availability, which can be studied in their native lipid environments.
Asunto(s)
Isótopos de Carbono/química , Hidrógeno/química , Proteínas de la Membrana/química , Resonancia Magnética Nuclear Biomolecular/métodos , ProtonesRESUMEN
Solid-state NMR (ssNMR) is a versatile technique that can provide high-resolution (sub-angstrom) structural data for integral membrane proteins embedded in native and model membrane environments. The methodologies for a priori structure determination have for the most part been developed using samples with crystalline and fibrous morphologies. However, the techniques are now being applied to large, polytopic membrane proteins including receptors, ion channels, and porins. ssNMR data may be used to annotate and refine existing structures in regions of the protein not fully resolved by crystallography (including ligand-binding sites and mobile solvent accessible loop regions). This review describes the spectroscopic experiments and data analysis methods (including assignment) used to generate high-resolution structural data for membrane proteins. We also consider the range of sample morphologies that are appropriate for study by this method.
Asunto(s)
Proteínas de la Membrana/química , Resonancia Magnética Nuclear Biomolecular/métodos , Sitios de Unión , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular/instrumentación , Conformación ProteicaRESUMEN
Over expression of proteins in E. coli frequently results in the production of inclusion bodies. Although ß(2) -microglobulin frequently forms fibrillar structures, our studies reveal significant differences between the protein in fibrils and inclusion bodies. This suggests that the formation of fibrils in inclusion bodies is dependent on the propensity of the protein to form fibrillar structures.