The transmembrane (TM) anchors of cell surface proteins had been one of the "blind spots" in structural biology because they are generally very hydrophobic and sometimes dynamics, and are thus difficult targets for structural characterization. A plethora of examples showed that these membrane anchors are not merely anchors but can multimerize specifically to activate signaling receptors on the cell surface or to stabilize the envelope proteins in viruses. Through a series of studies of the TM domains of immune receptors and viral membrane proteins, we have established a robust protocol for determining atomic resolution structures of TM oligomers by nuclear magnetic resonance (NMR) in bicelles that closely mimic a lipid bilayer. Here, we provide the details of the protocol consisting of five major sections: 1) general expression, purification, and bicelle reconstitution of hydrophobic TM and membrane-proximal domains; 2) determination of the oligomeric state of TM domains in bicelles; 3) detection of inter-molecular contacts; 4) structure determination; and 5) characterization of the protein TM partition. This protocol is broadly applicable to filling the structural gaps in membrane for many Type I/II membrane proteins.

After exposure to a doubled CO 2 concentration of 750 µ µ µ µ L L − − − − 1 for 2 months, average relative growth rate (RGR) of Mokara Yellow increased 25%. The two carboxylating enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPCase), responded differently to CO 2 enrichment. There was a significant daytime down-regulation in Rubisco activity in the leaves of CO 2 -enriched plants. However, PEPCase activity in CO 2 -enriched plants was much higher in the dark period, although it was slightly lower during the daytime than that at ambient CO 2 . Leaf sucrose-phosphate synthase (SPS) and sucrose synthase (SS) activities in CO 2 -enriched plants increased markedly, along with a night-time increase in total titratable acidity and malate accumulation. There was a remarkable increase in the levels of indole-3-acetic acid (IAA), gibberellins A 1 and A 3 (GA 1+3 ), isopentenyladenosine (iPA) and zeatin riboside (ZR) in the expanding leaves of plants grown at elevated CO 2 . It is suggested that (1) the down-regulation of Rubisco and upregulation of SPS and SS are two important acclimation processes that are beneficial because it enhanced both photosynthetic capacity at high CO 2 and reduced resource investment in excessive Rubisco capacity; (2) the increased levels of plant hormones in CO 2 -enriched M . Yellow might play an important role in controlling its growth and development.

This study demonstrated that immobilized Candida antarctica lipase B (N435) catalysis in bulk leads to higher molecular weight poly(glycerol sebacate), PGS, than self-catalyzed condensation polymerization. Since the glass-transition temperature, fragility, modulus, and strength for rubbery networks are inversely dependent on the concentration of chain ends, higher molecular weight PGS prepolymers will enable the preparation of cross-linked PGS matrices with unique mechanical properties. The evolution of molecular species during the prepolymerization step conducted at 120 °C for 24 h, prior to enzyme addition, revealed regular decreases in sebacic acid and glycerol-sebacate dimer with corresponding increases in oligomers with chain lengths from 3 to 7 units such that a homogeneous liquid substrate has resulted. At 67 h, for N435catalyzed PGS synthesis, the carboxylic acid conversion reached 82% without formation of a gel fraction, and number-average molecular weight (M n ) and weight-average molecular weight (M w ) values reached 6000 and 59 400 g/mol, respectively. In contrast, self-catalyzed PGS condensation polymerizations required termination at 55 h to avoid gelation, reached 72% conversion, and M n and M w values of 2600 and 13 800 g/mol, respectively. We also report the extent that solvent fractionation can enrich PGS in higher molecular weight chains. The use of methanol as a nonsolvent increased M n and M w by 131.7 and 18.3%, respectively, and narrower dispersity (Đ) decreased by 47.7% relative to the nonfractionated product.

apparent that conformational reorganization coupled to the ionization of the buried group is a major determinant of these pK a values. Specifically, the creation of charge in hydrophobic environments can trigger a shift from the fully folded state to local or partially unfolded states in which the charge can gain access to water or to an environment where the charge can be solvated. These alternative conformational states are not normally populated owing to the large free energy difference between the alternative and fully-folded native states; however, the partially unfolded states can become the new ground state under pH conditions where the internal group is charged. If the ionization of an internal group promotes the transition to a new conformational state then its pK a should be sensitive to the global thermodynamic stability (DG) of the protein because this determines the energy gap between the ground and the alternative states. This was tested by measuring the pK a of two internal Lys residues in variants of staphylococcal nuclease with thermodynamic stabilities ranging from 8.4 to 13.8 kcal/mol. The magnitude of the shift in the pK a of the internal Lys residues was found to be sensitive to the DG of the protein confirming that the pK a values of these Lys residues are determined by the probability of structural reorganization more than by local dielectric properties of their microenvironments. These observations imply that structure-based pK a calculations for buried groups and other electrostatic processes in hydrophobic environments require accurate treatment of conformational reorganization, which remains an extremely challenging proposition.

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Introduction The vascular endothelium is lined with the endothelial glycocalyx, composed of core proteins and glycosaminoglycans (GAGs). The loss of glycocalyx can cause endothelial injury and could contribute to the progression of retinopathy. The glycocalyx thickness has been shown to decrease in the diabetic retina; however, changes in individual components of the retinal glycocalyx have yet to be determined. Therefore, the present study examined hyperglycemia‐induced changes in retinal glycocalyx components. Methods Type‐1 diabetic rats and primary rat retinal microvascular endothelial cells (RRMECs) exposed to high glucose were used to mimic diabetes. Western blots and qRT‐PCR were used to analyze the expression of core proteins. Further, GAG composition was studied using LC‐MS. Results Both retinal endothelial cells and rat retina exhibited a significant decrease in mRNA transcripts and protein levels of syndecan‐3 under hyperglycemic condition, whereas syndecan‐1 expression was significantly increased. Further, we observed a substantial loss of glypican‐1 in RRMECs with high glucose, but a significant increase in the retina of diabetic rats. RRMECs in high glucose exhibited a significant decrease in chondroitin sulfate (CS) and hyaluronic acid (HA), but no change in heparan sulfate (HS) levels. In addition, 4‐O‐sulfated CS residues and 2‐O‐N‐sulfated HS residues were reduced, whereas N‐sulfated and 6‐O‐N‐sulfated HS residues were increased in retinal endothelial cells treated with high glucose. Lastly, we observed a significant increase in media levels of syndecan‐1, HA, HS, and CS, indicating increased shedding of glycocalyx components under hyperglycemia. Conclusions Our findings suggest that the loss of endothelial glycocalyx in the diabetic retina could be a result of a loss of the proteoglycan syndecan‐3 (and possibly glypican‐1) as well as the GAGs CS and HA.

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