Nanolipoprotein particles (NLPs) represent a unique nanometer-sized scaffold for supporting membrane proteins (MP). Characterization of their dynamic shape and association with MP in solution remains a challenge. Here, we present a rapid method of analysis by fluorescence correlation spectroscopy (FCS) to characterize bacteriorhodopsin (bR), a membrane protein capable of forming a NLP complex. By selectively labeling individual components of NLPs during cell-free synthesis, FCS enabled us to measure specific NLP diffusion times and infer size information for different NLP species. The resulting bR-loaded NLPs were shown to be dynamically discoidal in solution with a mean diameter of 7.8 nm. The insertion rate of bR in the complex was ~55% based on a fit model incorporating two separate diffusion properties to best approximate the FCS data. More importantly, based on these data, we infer that membrane protein associated NLPs are thermodynamically constrained as discs in solution, while empty NLPs appear to be less constrained and dynamically spherical.
Copyright © 2010 The Protein Society.lørdag 26. februar 2011
Characterizing diffusion dynamics of a membrane protein associated with nanolipoproteins using fluorescence correlation spectroscopy.
A secretory system for bacterial production of high-profile protein targets.
Escherichia coli represents a robust, inexpensive expression host for the production of recombinant proteins. However, one major limitation is that certain protein classes do not express well in a biologically relevant form using standard expression approaches in the cytoplasm of E. coli. To improve the usefulness of the E. coli expression platform we have investigated combinations of promoters a nd selected N-terminal fusion tags for the extracellular expression of human target proteins. A comparative study was conducted on 24 target proteins fused to outer membrane protein A (OmpA), outer membrane protein F (OmpF) and osmotically inducible protein Y (OsmY). Based on the results of this initial study we carried out an extended expression screen employing the OsmY fusion and multiple constructs of a more diverse set of human proteins. Using this high-throughput compatible system we clearly demonstrate that secreted biomedically relevant human proteins can be efficiently retrieved and purified from the growth medium.
Copyright © 2011 The Protein Society.fredag 25. februar 2011
Binding of small molecules to cavity forming mutants of a de novo designed protein.
A central goal of protein design is to devise novel proteins for applications in biotechnology and medicine. Many applications, including those focused on sensing and catalysis, will require proteins that recognize and bind to small molecules. Here we show that stably folded a-helical proteins isolated from a binary patterned library of designed sequences can be mutated to produce binding sites capable of binding a range of small aromatic compounds. Specifically, we mutated two phenylalanine side chains to alanine in the known structure of de novo protein S-824 to create buried cavities in the core of this 4-helix bundle. The parental protein and the Phe?Ala variants were exposed to mixtures of compounds, and selective binding was assessed by saturation transfer difference (STD) NMR. The affinities of benzene and a number of its derivatives were determined by pulse field gradient spin echo (PFGSE) NMR, and several of the compounds were shown to bind the mutated protein with micromolar dissociation constants. These studies suggest that stably folded de novo proteins from binary patterned libraries are well-suited as scaffolds for the design of binding sites.
Copyright © 2011 The Protein Society.torsdag 24. februar 2011
Crystal structure of the Nogo-receptor-2.
The inhibition of axon regeneration upon mechanical injury is dependent on interactions between Nogo receptors (NgRs) and their myelin-derived ligands. NgRs are composed of a leucine-rich repeat (LRR) region, thought to be structurally similar among the different isoforms of the receptor, and a divergent "stalk" region. It has been shown by others that the LRR and stalk regions of NgR1 and NgR2 have distinct roles in conferring binding affinity to the myelin associated glycoprotein (MAG) in vivo. Here we show that purified recombinant full length NgR1 and NgR2 maintain significantly higher binding affinity for purified MAG as compared to the isolated LRR region of either NgR1 or NgR2. We also present the crystal structure of the LRR and part of the stalk regions of NgR2 and compare it to the previously reported NgR1 structure with respect to the distinct signaling properties of the two receptor isoforms.
Copyright © 2011 The Protein Society.Crystal structure of a soluble form of human monoglyceride lipase in complex with an inhibitor at 1.35 A resolution.
A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2 arachidonoyl glycerol. A model is proposed in which monoglyceride lipase undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.
Copyright © 2011 The Protein Society.PMID: 21308848 [PubMed - as supplied by publisher]onsdag 23. februar 2011
Crystal structures of CmeR-bile acid complexes from Campylobacter jejuni.
The TetR family of transcription regulators are diverse proteins capable of sensing and responding to various structurally dissimilar antimicrobial agents. Upon detecting these agents, the regulators allow transcription of an appropriate array of resistance markers to counteract the deleterious compounds. Campylobacter jejuni CmeR is a pleiotropic regulator of multiple proteins, including the membrane-bound multidrug efflux transporter CmeABC. CmeR represses the expression of CmeABC and is induced by bile acids, which are substrates of the CmeABC tripartite pump. The multiligand-binding pocket of CmeR has been shown to be very extensive and consists of several positively charged and multiple aromatic amino acids. Here we describe the crystal structures of CmeR in complexes with the bile acids, taurocholate and cholate. Taurocholate and cholate are structurally related, differing by only the anionic charged group. However, these two ligands bind distinctly in the binding tunnel. Taurocholate spans the novel bile acid binding site adjacent to and without overlapping with the previously determined glycerol-binding site. The anionic aminoethanesulfonate group of taurocholate is neutralized by a charge-dipole interaction. Unlike taurocholate, cholate binds in an anti-parallel orientation but occupies the same bile acid-binding site. Its anionic pentanoate moiety makes a water-mediated hydrogen bond with a cationic residue to neutralize the formal negative charge. These structures underscore the promiscuity of the multifaceted binding pocket of CmeR. The capacity of CmeR to recognize bile acids was confirmed using isothermal titration calorimetry and fluorescence polarization. The results revealed that the regulator binds these acids with dissociation constants in the micromolar region.
Copyright © 2011 The Protein Society.Crystal structure of the sensory domain of Escherichia coli CadC, a member of the ToxR-like protein family.
The membrane-integral transcriptional activator CadC comprises sensory and transcriptional regulatory functions within one polypeptide chain. Its C-terminal periplasmic domain, CadC(pd) , is responsible for sensing of environmental pH as well as for binding of the feedback inhibitor cadaverine. Here we describe the crystal structure of CadC(pd) (residues 188-512) solved at a resolution of 1.8 Å via multiple wavelength anomalous dispersion (MAD) using a ReCl(6) (2-) derivative. CadC(pd) reveals a novel fold comprising two subdomains: an N-terminal subdomain dominated by a $\tilde \beta$sheet in contact with three a-helices and a C-terminal subdomain formed by a ten-membered a-helical bundle, which is oriented almost perpendicular to the helices in the first subdomain. Further to the native protein, crystal structures were also solved for its variants D471N and D471E, which show functionally different behavior in pH sensing. Interestingly, in the heavy metal derivative of CadC(pd) used for MAD phasing a ReCl(6) (2-) ion was found in a cavity located between the two subdomains. Amino acid side chains that coordinate this complex ion are conserved in CadC homologues from various bacterial species, suggesting a function of the cavity in the binding of cadaverine, which was supported by docking studies. Notably, CadC(pd) and its variants form dimers in solution, which can be explained by an extended, albeit rather polar interface between two symmetry-related monomers in the crystal structure. The occurrence of several acidic residues in this region suggests protonation-dependent changes in the mode of dimerization, which could eventually trigger transcriptional activation by CadC in the bacterial cytoplasm.
Copyright © 2011 The Protein Society.fredag 28. januar 2011
Curried Lentil Cashew Burgers
Curried Lentil Cashew Burgers
on November 15, 2010Recipe #441958
I got this from the Canadian Living Vegetarian Collection cookbook, and have added it to my "gold!" list of amazing vegetarian foods! Incredibly satisfying and loaded with the good stuff, these burgers are a wonderful meal for everyone. Try it, I guarantee you won't be sorry!
Serving Size: 1 (203 g)
Servings Per Recipe: 4
Amount Per Serving% Daily ValueCalories 572.2 Calories from Fat 14325%Total Fat 15.9 g24%Saturated Fat 2.4 g12%Monounsaturated Fat 6.3 g31%Polyunsaturated Fat 5.7 g28%Trans Fat 0.0 g0%Cholesterol 0.0 mg0%Sodium 57.8 mg2%Potassium 1293.9 mg36%Magnesium 184.0 mg7%Total Carbohydrate 77.1 g25%Dietary Fiber 34.6 g138%Sugars 4.7 g19%Protein 32.2 g64%torsdag 27. januar 2011
Simple Turkey Meatloaf
Simple Turkey Meatloaf
on November 15, 2010Recipe #442000
Well seasoned, moist, hearty turkey meatloaf that even a child could make,
Serving Size: 1 (205 g)
Servings Per Recipe: 6
Amount Per Serving% Daily ValueCalories 489.2 Calories from Fat 20341%Total Fat 22.6 g34%Saturated Fat 6.3 g31%Monounsaturated Fat 8.4 g42%Polyunsaturated Fat 5.1 g25%Trans Fat 0.6 g0%Cholesterol 285.6 mg95%Sodium 1837.0 mg76%Potassium 808.7 mg23%Magnesium 75.2 mg3%Total Carbohydrate 23.4 g7%Dietary Fiber 0.9 g3%Sugars 8.2 g33%Protein 46.2 g92%Salvadorian Roasted Turkey
Salvadorian Roasted Turkey
on November 15, 2010Recipe #441990
This is how my mother-in-law makes her turkey. It was definitely a change from my very traditional Canadian turkey. however, the flavor combinations are amazing and this recipe makes an amazingly juicy turkey.
Serving Size: 1 (396 g)
Servings Per Recipe: 12
Amount Per Serving% Daily ValueCalories 542.6 Calories from Fat 26348%Total Fat 29.3 g45%Saturated Fat 7.3 g36%Monounsaturated Fat 10.8 g54%Polyunsaturated Fat 8.3 g41%Trans Fat 0.0 g0%Cholesterol 188.1 mg62%Sodium 369.7 mg15%Potassium 1052.0 mg30%Magnesium 105.8 mg4%Total Carbohydrate 8.5 g2%Dietary Fiber 2.6 g10%Sugars 3.6 g14%Protein 59.1 g118%