A survey of detergents for the purification of stable,active human cystic fibrosis transmembrane conductance regulator (CFTR)

Structural knowledge of the cystic fibrosis transmembrane conductance regulator (CFTR) requires developing methods to purify and stabilize this aggregation-prone membrane protein above 1 mg/ml. Starting with green fluorescent protein- and epitope-tagged human CFTR produced in mammalian cells known to properly fold and process CFTR, we devised a rapid tandem affinity purification scheme to minimize CFTR exposure to detergent in order to preserve its ATPase function. We compared a panel of detergents, including widely used detergents (maltosides, neopentyl glycols (MNG), C₁₂E₈, lysolipids, Chaps) and innovative detergents (branched alkylmaltosides, facial amphiphiles) for CFTR purification, function, monodispersity and stability. ATPase activity after reconstitution into proteoliposomes was 2–3 times higher when CFTR was purified using facial amphiphiles. ATPase activity was also demonstrated in purified CFTR samples without detergent removal using a novel lipid supplementation assay. By electron microscopy, negatively stained CFTR samples were monodisperse at low concentration, and size exclusion chromatography showed a predominance of monomer even after CFTR concentration above 1 mg/ml. Rates of CFTR aggregation quantified in an electrophoretic mobility shift assay showed that detergents which best preserved reconstituted ATPase activity also supported the greatest stability, with CFTR monomer half-lives of 6–9 days in MNG or Chaps, and 12–17 days in facial amphiphile. Cryoelectron microscopy of concentrated CFTR in MNG or facial amphiphile confirmed mostly monomeric protein, producing low resolution reconstructions in conformity with similar proteins. These protocols can be used to generate samples of pure, functional, stable CFTR at concentrations amenable to biophysical characterization.     

Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains

Detergent interaction with extramembranous soluble domains (ESDs) is not commonly considered an important determinant of integral membrane protein (IMP) behavior during purification and crystallization, even though ESDs contribute to the stability of many IMPs. Here we demonstrate that some generally nondenaturing detergents critically destabilize a model ESD, the first nucleotide‐binding domain (NBD1) from the human cystic fibrosis transmembrane conductance regulator (CFTR), a model IMP. Notably, the detergents show equivalent trends in their influence on the stability of isolated NBD1 and full‐length CFTR. We used differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy to monitor changes in NBD1 stability and secondary structure, respectively, during titration with a series of detergents. Their effective harshness in these assays mirrors that widely accepted for their interaction with IMPs, i.e., anionic > zwitterionic > nonionic. It is noteworthy that including lipids or nonionic detergents is shown to mitigate detergent harshness, as will limiting contact time. We infer three thermodynamic mechanisms from the observed thermal destabilization by monomer or micelle: (i) binding to the unfolded state with no change in the native structure (all detergent classes); (ii) native state binding that alters thermodynamic properties and perhaps conformation (nonionic detergents); and (iii) detergent binding that directly leads to denaturation of the native state (anionic and zwitterionic). These results demonstrate that the accepted model for the harshness of detergents applies to their interaction with an ESD. It is concluded that destabilization of extramembranous soluble domains by specific detergents will influence the stability of some IMPs during purification.     

Heparan sulfate chains of syndecan-1 regulate ectodomain shedding

Matrix metalloproteinases release intact syndecan-1 ectodomains from the cell surface giving rise to a soluble, shed form of the proteoglycan. Although it is known that shed syndecan-1 controls diverse pathophysiological responses in cancer, wound healing, inflammation, infection, and immunity, the mechanisms regulating shedding remain unclear. We have discovered that the heparan sulfate chains present on syndecan core proteins suppress shedding of the proteoglycan. Syndecan shedding is dramatically enhanced when the heparan sulfate chains are enzymatically degraded or absent from the core protein. Exogenous heparan sulfate or heparin does not inhibit shedding, indicating that heparan sulfate must be attached to the core protein to suppress shedding. Regulation of shedding by heparan sulfate occurs in multiple cell types, for both syndecan-1 and syndecan-4 and in murine and human syndecans. Mechanistically, the loss of heparan sulfate enhances the susceptibility of the core protein to proteolytic cleavage by matrix metalloproteinases. Enhanced shedding of syndecan-1 following loss of heparan sulfate is accompanied by a dramatic increase in core protein synthesis. This suggests that in response to an increase in the rate of shedding, cells attempt to maintain a significant level of syndecan-1 on the cell surface. Together these data indicate that the amount of heparan sulfate present on syndecan core proteins regulates both the rate of syndecan shedding and core protein synthesis. These findings assign new functions to heparan sulfate chains, thereby broadening our understanding of their physiological importance and implying that therapeutic inhibition of heparan sulfate degradation could impact the progression of some diseases.     

3-methyladenine-DNA glycosylase II: the crystal structure of an AlkA-hypoxanthine complex suggests the possibility of product inhibition

Escherichia coli (E. coli) protein 3-methyladenine-DNA glycosylase II (AlkA) functions primarily by removing alkylation damage from duplex and single stranded DNA. A crystal structure of AlkA was refined to 2.0 A resolution. This structure in turn was used to refine an AlkA-hypoxanthine (substrate) complex structure to 2.4 A resolution. The complex structure shows hypoxanthine located in AlkA's active site stacked between residues W218 and Y239. The structural analysis of the AlkA and AlkA-hypoxanthine structures indicate that free hypoxanthine binding in the active site may inhibit glycosylase activity.     

Diversity among bacteria isolated from the deep subsurface

Culturable bacteria from the deep subsurface (179 m) at Cerro Negro, New Mexico were isolated and characterized. The average number of viable aerobic bacteria was estimated to be 5×10⁵g^–1 of sediment, but only about 0.1% of these could be recovered on agar medium when incubated under aerobic conditions. Of 158 strains isolated from this depth, 92 were characterized by cellular fatty acid profiles (FAME), 36 by analysis of partial 16S rDNA sequences, and 44 by rep-PCR genome fingerprint analysis using three different sets of oligonucleotide primers (REP, BOX, or ERIC). These analyses showed the majority of isolates (67%) were Gram-positive bacteria and primarily members of genera with a high %G+C DNA. The remaining isolates were -subdivisionProteobacteria (19%) and members of the flavobacteria group (14%). The diversity indices based on these different methods of characterization were very high suggesting this subsurface habitat harbors a highly diverse microbial community.     

Stimulation of Growth and Nucleic Acid Biosynthesis at Low Concentration of Abscisic Acid in Tissue Culture of Spinacia oleracea

The effect of abscisic acid on growth, ultrastructure and nucleic acid biosynthesis was studied in tissue culture of spinach (Spinacia oleracea L.). Low concentration (0.01 mg l^?1) of abscisic acid increased fresh and dry weight of calluses, whereas 1.0 mg l^?1 was inhibitory. The stimulating effect was observed only in the presence of a relatively high concentration of kinetin (1 mg l^?1). The inhibitory effect was partly overcome by the same kinetin concentration. The low concentration of abscisic acid probably accelerated the induction of callus growth after subculture and stimulated cell division in the exponential phase of growth. Electron microscopy showed the presence of numerous polysomes and rough endoplasmic reticulum in callus cells grown at the stimulating abscisic acid concentration. Control cells and cells at the inhibitory concentration had slightly hyaline cytoplasm and were more vacuolated. Incubation of callus tissue with ³²P in the presence of stimulating concentration of abscisic acid showed a significant increase in the rate of biosynthesis of all nucleic acid classes after 8 h, whereas inhibitory concentration produced a decrease in ³²P incorporation. However, when the tissue was grown in the presence of abscisic acid for 20 days, both concentrations decreased the rate of nucleic acid biosynthesis, as compared to the controls.