SPR and ITC determination of the kinetics and the thermodynamics of bivalent versus monovalent sugar ligand–lectin interactions

A kinetic study of the interaction of bivalent and monovalent sugar ligands with a lectin was undertaken with the aid of surface plasmon resonance (SPR) method. The study involved a series of bivalent α-d-mannopyranoside containing sugar ligands, with systematic variation in the distance between the sugar ligands. The detailed kinetic studies showed that bivalent ligands underwent a faster association (k _on) and a slower dissociation (k _off) of the ligand–lectin complexes, in comparison to the monovalent ligand–lectin complexes. The kinetic constants were complemented further by assessing the thermodynamic parameters with the aid of isothermal titration calorimetry (ITC). The initiation of cross-linking of ligand–lectin interactions emerge from the early stages of the complexation. The dynamic light scattering (DLS) and the transmission electron microscopy (TEM) techniques allowed judging the sizes and morphologies of the complex in the solution and solid states, respectively.     

Dipolar assisted rotational resonance NMR of tryptophan and tyrosine in rhodopsin

Two dimensional (2D) solid-state (13)C.(13)C dipolar recoupling experiments are performed on a series of model compounds and on the visual pigment rhodopsin to establish the most effective method for long range distance measurements in reconstituted membrane proteins. The effects of uniform labeling, inhomogeneous B(1) fields, relaxation and dipolar truncation on cross peak intensity are investigated through NMR measurements of simple amino acid and peptide model compounds. We first show that dipolar assisted rotational resonance (DARR) is more effective than RFDR in recoupling long-range dipolar interactions in these model systems. We then use DARR to establish (13)C-(13)C correlations in rhodopsin. In rhodopsin containing 4'-(13)C-Tyr and 8,19-(13)C retinal, we observe two distinct tyrosine-to-retinal correlations in the DARR spectrum. The most intense cross peak arises from a correlation between Tyr268 and the retinal 19-(13)CH(3), which are 4.8 A apart in the rhodopsin crystal structure. A second cross peak arises from a correlation between Tyr191 and the retinal 19-(13)CH(3), which are 5.5 A apart in the crystal structure. These data demonstrate that long range (13)C em leader (13)C correlations can be obtained in non-crystalline integral membrane proteins reconstituted into lipid membranes containing less than 150 nmoles of protein. In rhodopsin containing 2-(13)C Gly121 and U-(13)C Trp265, we do not observe a Trp-Gly cross peak in the DARR spectrum despite their close proximity (3.6 A) in the crystal structure. Based on model compounds, the absence of a (13)C em leader (13)C cross peak is due to loss of intensity in the diagonal Trp resonances rather than to dipolar truncation.     

CADB: Conformation Angles DataBase of proteins

Conformation Angles DataBase (CADB) provides an online resource to access data on conformation angles (both main-chain and side-chain) of protein structures in two data sets corresponding to 25% and 90% sequence identity between any two proteins, available in the Protein Data Bank. In addition, the database contains the necessary crystallographic parameters. The package has several flexible options and display facilities to visualize the main-chain and side-chain conformation angles for a particular amino acid residue. The package can also be used to study the interrelationship between the main-chain and side-chain conformation angles. A web based JAVA graphics interface has been deployed to display the user interested information on the client machine. The database is being updated at regular intervals and can be accessed over the World Wide Web interface at the following URL: http://144.16.71.148/cadb/.     

Development of a large-scale biocalorimeter to monitor and control bioprocesses

Calorimetry has shown real potential at bench-scale for chemical and biochemical processes. The aim of this work was therefore to scale-up the system by adaptation of a standard commercially available 300-L pilot-scale bioreactor. To achieve this, all heat flows entering or leaving the bioreactor were identified and the necessary instrumentation implemented to enable on-line monitoring and dynamic heat balance estimation. Providing that the signals are sufficiently precise, such a heat balance would enable calculation of the heat released or taken up during an operational (bio)process. Two electrical Wattmeters were developed, the first for determination of the power consumption by the stirrer motor and the second for determination of the power released by an internal calibration heater. Experiments were designed to optimize the temperature controller of the bioreactor such that it was sufficiently rapid so as to enable the heat accumulation terms to be neglected. Further calibration experiments were designed to correlate the measured stirring power to frictional heat losses of the stirrer into the reaction mass. This allows the quantitative measurement of all background heat flows and the on-line quantitative calculation of the (bio)process power. Three test fermentations were then performed with B. sphaericus 1593M, a spore-forming bacterium pathogenic to mosquitoes. A first batch culture was performed on a complex medium, to enable optimization of the calorimeter system. A second batch culture, on defined medium containing three carbon sources, was used to show the fast, accurate response of the heat signal and the ability to perfectly monitor the different growth phases associated with growth on mixed substrates, in particular when carbon sources became depleted. A maximum heat output of 1100 W was measured at the end of the log-phase. A fed-batch culture on the same defined medium was then carried out with the feed rate controlled as a function of the calorimeter signal. A maximum heat output of 2250 W was measured at the end of the first log-phase. This work demonstrates that real-time quantitative calorimetry is not only possible at pilot-scale, but could be readily applied at even larger scales. The technique requires simple, readily available devices for determination of the few necessary heat flows, making it a robust, cost-effective technique for process development and routine monitoring and control of production processes.     

Functional Complementation of Nontoxic Mutant Binary Toxins of Bacillus sphaericus 1593M Generated by Site-Directed Mutagenesis

Alanine residues were substituted by site-directed mutagenesis at selected sites of the N- and C-terminal regions of the binary toxin (51- and 42-kDa peptides) of B. sphaericus 1593M, and the mutant toxins were cloned and expressed in Escherichia coli. Bioassays with mosquito larvae, using binary toxins derived from individual mutants, showed that the substitution of alanine at some sites in both the 51-kDa and the 42-kDa peptides resulted in a total loss of activity. Surprisingly, after mixing two nontoxic derivatives of the same peptide, i.e., one mutated at the N-terminal end and the other mutated at the C-terminal end of either the 51-kDa or the 42-kDa peptide, the toxicity was restored. This result indicates that the altered binary toxins can functionally complement each other by forming oligomers.