Native Conformation at Specific Residues in Recombinant Inclusion Body Protein in Whole Cells Determined with Solid-State NMR Spectroscopy

Curtis-Fisk, Jaime; Spencer, Ryan M.; Weliky, David P.

doi:10.1021/ja8039426

Cited by 50 publications

(41 citation statements)

References 21 publications

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“…Even in the presence of considerable molecular motion, it is possible for residual anisotropies to exist as in the case of liquid crystals [81–83]. As a result, the magnetic or electrical interactions in NMR spectroscopy provide a rich source of information about structure and dynamics for membrane proteins [21, 84–94] and peptides [95–103], lipid bilayers [104–108], biopolymer fibers [109, 110], amyloid fibrils [111–118], and inclusion bodies [119]. …”

Section: Solid-state Nmr Spectroscopy Is a Powerful Tool In Membramentioning

confidence: 99%

Retinal dynamics during light activation of rhodopsin revealed by solid-state NMR spectroscopy

Brown

Salgado

Struts

2010

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Rhodopsin is a canonical member of class A of the G protein-coupled receptors (GPCR) that are implicated in many of the drug interventions in humans and are of great pharmaceutical interest. The molecular mechanism of rhodopsin activation remains unknown as atomistic structural information for the active metarhodopsin II state is currently lacking. Solid-state 2H NMR constitutes a powerful aproarch to study atomic-level dynamics of membrane proteins. In the present application we describe how information is obtained about interactions of the retinal cofactor with rhodopsin that change with light activation of the photoreceptor. The retinal methyl groups play an important role in rhodopsin function by directing conformational changes upon transition into the active state. Site-specific 2H labels have been introduced into the methyl groups of retinal and solid-state 2H NMR methods applied to obtain order parameters and correlation times that quantify the mobility of the cofactor in the inactive dark state, as well as the cryo-trapped metarhodopsin I and metarhodopsin II states. Analysis of the angular-dependent 2H NMR lineshapes for selectively deuterated methyl groups of rhodopsin in aligned membranes enables determination of the average ligand conformation within the binding pocket. The relaxation data suggest that the β-ionone ring is not expelled from its hydrophobic pocket in the transition from the pre-activated metarhodopsin I to the active metarhodopsin II state. Rather, the major structural changes of the retinal cofactor occur already at the metarhodopsin I state in the activation process. The metarhodopsin I to metarhodopsin II transition involves mainly conformational changes of the protein within the membrane lipid bilayer rather than the ligand. The dynamics of the retinylidene methyl groups upon isomerization are explained by an activation mechanism involving cooperative rearrangments of extracellular loop E2 together with transmembrane helices H5 and H6. These activating movements are triggered by steric clashes of the isomerized all-trans retinal with the β4 strand of the E2 loop and the side chains of Glu122 and Trp265 within the binding pocket. The solid-state 2H NMR data are discussed with regard to the pathway of the energy flow in the receptor activation mechanism.

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Section: Solid-state Nmr Spectroscopy Is a Powerful Tool In Membramentioning

confidence: 99%

Retinal dynamics during light activation of rhodopsin revealed by solid-state NMR spectroscopy

Brown

Salgado

Struts

2010

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…Thus, despite the prevalent roles that biofilms play in human infection, there has been little reliable quantitative information available regarding biofilm matrix composition. Solid-state NMR spectroscopy is uniquely suited to study complex and insoluble biological materials (12) such as whole cells (13)(14)(15)(16)(17)(18), biofilms, and the extracellular matrix (19)(20)(21)(22). Solidstate NMR approaches can be used to quantify composition and to measure internuclear distances to determine parameters of architecture in such macromolecular assemblies and does not require soluble or crystalline samples (15).…”

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Analysis of the Aspergillus fumigatus Biofilm Extracellular Matrix by Solid-State Nuclear Magnetic Resonance Spectroscopy

et al. 2015

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dAspergillus fumigatus is commonly responsible for lethal fungal infections among immunosuppressed individuals. A. fumigatus forms biofilm communities that are of increasing biomedical interest due to the association of biofilms with chronic infections and their increased resistance to antifungal agents and host immune factors. Understanding the composition of microbial biofilms and the extracellular matrix is important to understanding function and, ultimately, to developing strategies to inhibit biofilm formation. We implemented a solid-state nuclear magnetic resonance (NMR) approach to define compositional parameters of the A. fumigatus extracellular matrix (ECM) when biofilms are formed in RPMI 1640 nutrient medium. Whole biofilm and isolated matrix networks were also characterized by electron microscopy, and matrix proteins were identified through protein gel analysis. The 13 C NMR results defined and quantified the carbon contributions in the insoluble ECM, including carbonyls, aromatic carbons, polysaccharide carbons (anomeric and nonanomerics), aliphatics, etc. Additional 15 N and 31 P NMR spectra permitted more specific annotation of the carbon pools according to C-N and C-P couplings. Together these data show that the A. fumigatus ECM produced under these growth conditions contains approximately 40% protein, 43% polysaccharide, 3% aromatic-containing components, and up to 14% lipid. These fundamental chemical parameters are needed to consider the relationships between composition and function in the A. fumigatus ECM and will enable future comparisons with other organisms and with A. fumigatus grown under alternate conditions. A spergillus fumigatus is the most important etiological agent of human aspergillosis and is recognized as a highly allergenic and opportunistic pathogen, causing acute and chronic infections particularly among immunocompromised individuals (1). Hospital-associated outbreaks of A. fumigatus may occur during periods of construction or renovation (2) or when fungi colonize water distribution systems that then lead to spore aerosolization in patient care areas and patient exposure (1). Additionally, A. fumigatus can cause infections as a result of colonizing medical implant devices, including cardiac pacemakers, joint replacements, and breast implants (3). Despite improvements in diagnostics and the antifungal armamentarium, including broad-spectrum azoles and the echinocandin antifungals, mortality due to A. fumigatus invasive infections remains high.Like many microorganisms, A. fumigatus can also assemble into multicellular communities, termed biofilms, composed of cells plus an extracellular matrix (ECM) (4-9). Although more work is needed to fully understand the functional implications of biofilm formation by A. fumigatus, recent evidence suggests that the ECM may provide the infrastructure for enhancing cell density, controlling disaggregation, and altering nutritional needs, as well as providing a protective physical and chemical barrier that can decrease sensitivity to competi...

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“…It has been suggested that though aggregated, the protein in the inclusion bodies may be in its native conformation and functionally active (10,14,17). In these cases, the protein is easier to recover since the overall tertiary fold of the protein is not lost during aggregation.…”

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confidence: 99%

Effect of Chemical Chaperones in Improving the Solubility of Recombinant Proteins in Escherichia coli

Prasad

Khadatare

Roy

2011

Appl Environ Microbiol

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The recovery of active proteins from inclusion bodies usually involves chaotrope-induced denaturation, followed by refolding of the unfolded protein. The efficiency of renaturation is low, leading to reduced yield of the final product. In this work, we report that recombinant proteins can be overexpressed in the soluble form in the host expression system by incorporating compatible solutes during protein expression. Green fluorescent protein (GFP), which was otherwise expressed as inclusion bodies, could be made to partition off into the soluble fraction when sorbitol and arginine, but not ethylene glycol, were present in the growth medium. Arginine and sorbitol increased the production of soluble protein, while ethylene glycol did not. Production of ATP increased in the presence of sorbitol and arginine, but not ethylene glycol. A control experiment with fructose addition indicated that protein solubilization was not due to a simple ATP increase. We have successfully reproduced these results with the N-terminal domain of HypF (HypF-N), a bacterial protein which forms inclusion bodies in Escherichia coli. Instead of forming inclusion bodies, HypF-N could be expressed as a soluble protein in the presence of sorbitol, arginine, and trehalose in the expression medium.Overexpression of proteins in heterologous systems often results in the formation of inclusion bodies. These electronrefractile particles are formed because of the failure of prokaryotic folding machinery to correctly fold the nascent polypeptide chain, increasing its local concentration in the cytosol. The most common strategy to recover active proteins from these particles is by denaturing them in the presence of chaotropes and refolding them to the native conformation by the gradual removal of the denaturant. This process results in high loss in protein yield since the renaturation efficiency of most proteins is not very high.

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Native Conformation at Specific Residues in Recombinant Inclusion Body Protein in Whole Cells Determined with Solid-State NMR Spectroscopy

Cited by 50 publications

References 21 publications

Retinal dynamics during light activation of rhodopsin revealed by solid-state NMR spectroscopy

Retinal dynamics during light activation of rhodopsin revealed by solid-state NMR spectroscopy

Analysis of the Aspergillus fumigatus Biofilm Extracellular Matrix by Solid-State Nuclear Magnetic Resonance Spectroscopy

Effect of Chemical Chaperones in Improving the Solubility of Recombinant Proteins in Escherichia coli

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