Chimeric β-Lactamases: Global Conservation of Parental Function and Fast Time-Scale Dynamics with Increased Slow Motions

Enzyme engineering has been facilitated by recombination of close homologues, followed by functional screening. In one such effort, chimeras of two class-A β-lactamases – TEM-1 and PSE-4 – were created according to structure-guided protein recombination and selected for their capacity to promote bacterial proliferation in the presence of ampicillin (Voigt et al., Nat. Struct. Biol. 2002 9:553). To provide a more detailed assessment of the effects of protein recombination on the structure and function of the resulting chimeric enzymes, we characterized a series of functional TEM-1/PSE-4 chimeras possessing between 17 and 92 substitutions relative to TEM-1 β-lactamase. Circular dichroism and thermal scanning fluorimetry revealed that the chimeras were generally well folded. Despite harbouring important sequence variation relative to either of the two ‘parental’ β-lactamases, the chimeric β-lactamases displayed substrate recognition spectra and reactivity similar to their most closely-related parent. To gain further insight into the changes induced by chimerization, the chimera with 17 substitutions was investigated by NMR spin relaxation. While high order was conserved on the ps-ns timescale, a hallmark of class A β-lactamases, evidence of additional slow motions on the µs-ms timescale was extracted from model-free calculations. This is consistent with the greater number of resonances that could not be assigned in this chimera relative to the parental β-lactamases, and is consistent with this well-folded and functional chimeric β-lactamase displaying increased slow time-scale motions.     

Structural basis of the preferential binding for globo-series glycosphingolipids displayed by Pseudomonas aeruginosa lectin I

The opportunistic pathogen Pseudomonas aeruginosa contains several carbohydrate-binding proteins, among which is the P. aeruginosa lectin I (PA-IL), which displays affinity for alpha-galactosylated glycans. Glycan arrays were screened and demonstrated stronger binding of PA-IL toward alphaGal1-4betaGal-terminating structures and weaker binding to alphaGal1-3betaGal ones in order to determine which human glycoconjugates could play a role in the carbohydrate-mediated adhesion of the bacteria. This was confirmed in vivo by testing the binding of the lectin to Burkitt lymphoma cells that present large amounts of globotriaosylceramide antigen Gb3/CD77/P(k). Trisaccharide moieties of Gb3 (alphaGal1-4betaGal1-4Glc) and isoglobotriaosylceramide (alphaGal1-3betaGal1-4Glc) were tested by titration microcalorimetry, and both displayed similar affinity to PA-IL in solution. The crystal structure of PA-IL complexed to alphaGal1-3betaGal1-4Glc trisaccharide has been solved at 1.9-A resolution and revealed how the second galactose residue makes specific contacts with the protein surface. Molecular modeling studies were performed in order to compare the binding mode of PA-IL toward alphaGal1-3Gal with that toward alphaGal1-4Gal. Docking studies demonstrated that alphaGal1-4Gal creates another network of contacts for achieving a very similar affinity, and 10-ns molecular dynamics in explicit water allowed for analyzing the flexibility of each disaccharide ligand in the protein binding site. The higher affinity observed for binding to Gb3 epitope, both in vivo and on glycan array, is likely related to the presentation effect of the oligosaccharide on a surface, since only the Gb3 glycosphingolipid geometry is fully compatible with parallel insertion of neighboring trisaccharide heads in two binding sites of the same tetramer of PA-IL.     

Chimeric fusions of subunit IV and PetL in the b6f complex of Chlamydomonas reinhardtii: structural implications and consequences on state transitions.

The cytochrome b(6)f complex of Chlamydomonas reinhardtii contains four large subunits and at least three small ones, PetG, PetL, and PetM, whose role and location are unknown. Chimeric proteins have been constructed, in which the C terminus of subunit IV is fused to either one or the other of the two putative N termini of PetL. Biochemical and functional analysis of the chimeras together with mass spectrometry analysis of the wild-type (WT) complex led to the following conclusions: (i) neither a free subunit IV C terminus nor a free PetL N terminus is required for assembly of the b(6)f complex; (ii) the first AUG codon in the sequence of the gene petL is used for initiation; (iii) the N terminus of WT PetL lies in the lumen; (iv) in the WT complex, the N terminus of PetL and the C terminus of subunit IV are within reach of each other; (v) the purified b(6)f complex from C. reinhardtii contains an eighth, hitherto unrecognized subunit, PetN; and (vi) the ability to perform state transitions is lost in the chimeric mutants, although (vii) the Q-cycle is unaffected. A structural hypothesis is presented to account for this peculiar phenotype.     

Glycolipid-Enriched Membrane Domains Are Assembled into Membrane Patches by Associating with the Actin Cytoskeleton

Nonionic detergent lysates of cells contain a glycolipid-enriched membrane (GEM) fraction. It has been proposed that the GEM fraction represents poorly solubilized GEM microdomains, or lipid rafts. However, the properties of GEM domains in intact cells remain controversial. To study the properties of a GEM-associated protein using confocal microscopy, GFP was targeted to GEM domains using the N-terminal domain of p56(lck) (LckNT). Imaging of HeLa cells expressing LckNT-GFP showed that it was targeted to large actin-rich patches in the plasma membrane that contained up to a fivefold enrichment of protein. Double-labeling experiments showed that the patches were selectively enriched with other GEM-associated molecules. Furthermore, the patches were resistant to extraction by TX-100, and disrupting GEM domains by extracting cholesterol also disrupted colocalization of LckNT-GFP with F-actin. Analogous to the actin-rich patches in HeLa cells, LckNT-GFP colocalized with actin-rich membrane caps in stimulated T cells. Furthermore, disrupting the GEM-targeting signal of LckNT-GFP also inhibited its targeting to membrane caps. Altogether, these findings extend previous studies by showing that association of GEM domains with the actin cytoskeleton provides a mechanism for targeting signaling molecules to membrane patches and caps.     

Formation of insulin-secreting, Sertoli-enriched tissue constructs by microgravity coculture of isolated pig islets and rat Sertoli cells

Pancreatic islets, isolated from neonatal pigs, and Sertoli cells, isolated from prepubertal rats, were cocultured in simulated microgravity utilizing the NASA-developed highly accelerating, rotating vessel (HARV) biochamber. Following 5 d of incubation, three-dimensional Sertoli-islet cell aggregates (SICA) retained the ability to secrete insulin when exposed to elevated glucose. SICA contained FasL-positive Sertoli cells and insulin-positive beta-cells randomly organized within the spherical construct. The addition of 1% Matrigel induced the reorganization of aggregates (SICAs formed in the presence of Matrigel [SICAmgs]) showing the peripherialization and epithelialization of Sertoli cells and the centralization of islets in association with lumen-like spaces. The Sertoli cells, but not Matrigel, aided in preserving the structural integrity of HARV-incubated islets. Neither Matrigel nor Sertoli cells appeared to interfere with the ability of SICA or SICA mg to secrete insulin and express FasL.     

Increased abundance of the non-indigenous zooplanktivore, <Emphasis Type="Italic">Bythotrephes longimanus</Emphasis>, is strongly correlated with greater spring prey availability in Canadian Shield lakes

The non-indigenous zooplanktivore, Bythotrephes longimanus, is a large Palaearctic cladoceran that is spreading rapidly in the Great Lakes watershed in North America. As a voracious predator, Bythotrephes can reduce herbivorous cladoceran abundance and diversity; however, the variables that affect its abundance are not well understood. To determine what bottom-up factors are associated with the abundance and seasonal dynamics of established Bythotrephes populations, two Bythotrephes datasets from lakes in south-central Ontario, Canada, were analysed using multiple regression and multivariate analyses: a multi-lake dataset of nine lakes sampled in 2003 and a multi-year dataset of one of these lakes, Harp Lake, sampled from 1994–1998 and 2001–2004. Bottom-up variables tested were Secchi disk depth, epilimnetic temperature, cladoceran (prey) density, total phosphorus, dissolved organic carbon and Chlorophyll a, as well as maximum depth for the multi-lake dataset. In both analyses and datasets, springtime abundance of herbivorous cladocerans was consistently found to be a significant factor associated with Bythotrephes (June–September) abundance; Bythotrephes annual abundance was significantly and positively associated with mean May and June prey abundance, along with mean Secchi disk depth for the multi-lake dataset, and groups of lakes or years with similar Bythotrephes seasonal abundance patterns were predicted by June prey abundance. Additionally, prey availability was the dominant contributor towards changes in weekly Bythotrephes birth rates calculated for two of the study lakes. Our study suggests that prey availability influences Bythotrephes abundance, which provides evidence that Bythotrephes establishment success is affected by the abundance of its prey.     

Ultrathin-layer microelectrophoretic determination of lactate dehydrogenase isoenzymes in corneal and conjunctival epithelium of the cow

In order to evaluate the impact of tissue oxygenation on the distribution pattern of lactate dehydrogenase isoenzymes, activities of the isoenzymes were measured in microdissected samples of bovine tissue. A highly sensitive ultrathin-layer electrophoretic technique was used to determine the distribution pattern of lactate dehydrogenase isoenzymes in basal, intermediate and superficial layers of the epithelium of central and peripheral cornea and in the epithelium of the bulbar conjunctiva. Measurements revealed almost homogeneous intraepithelial distribution patterns of lactate dehydrogenase isoenzymes in both tissues. In the cornea the lactate dehydrogenase isoenzymes 4 and 5, which are regarded to be specialized for anaerobic glucose metabolism, were found to predominate. In the well-oxygenated conjunctival epithelium most of the activity could be ascribed to the lactate dehydrogenase isoenzyme 3. In contrast to the isoenzymatic activities, total activity of lactate dehydrogenase was inhomogeneously distributed; maximum activities were found in the basal layer of corneal epithelium and in the intermediate layer of conjunctival epithelium. The results indicate that oxygen supply is relevant rather for the intraepithelial distribution of total enzyme activity than for the expression of lactate dehydrogenase isoenzymes.Parts of this study were presented as an inaugural dissertation to the Medical Faculty of the University of Basel by K. Krieger