Development of monoclonal antibodies for monitoring Aedes atropalpus vitellogenesis

Monoclonal antibodies to a mixture of Aedes atropalpus and A. aegypti soluble yolk proteins were produced by hybridomas between the fusion of P3X63.653 myeloma cells and splenocytes of immunized BALB/c mice. Ascites fluid collected from mice innoculated with cloned hybridoma cells contained high specificity and affinity to the soluble yolk proteins of both Aedes species. Seven different hybridoma lines produced antibodies with specificity to both A. atropalpus and A. aegypti and one cell line produced antibodies monospecific to A. aegypti soluble yolk proteins. Monoclonal antibodies specific to A. atropalpus vitellin and vitellogenin were characterized by a combination of gel electrophoresis, western blotting and immunohistochemical staining. An indirect double antibody sandwich enzyme-linked immunosorbent assay was developed using a mixture of the seven hybridoma antibodies to A. atropalpus vitellin for monitoring vitellogenin levels in individual mosquito haemolymph samples. With this procedure, the peak period of vitellogenin synthesis in A. atropalpus was found to be 18 to 30 h after adult eclosion.     

The Center for Optimized Structural Studies (COSS) platform for automation in cloning,expression, and purification of single proteins and protein–protein complexes

Expression in Escherichia coli represents the simplest and most cost effective means for the production of recombinant proteins. This is a routine task in structural biology and biochemistry where milligrams of the target protein are required in high purity and monodispersity. To achieve these criteria, the user often needs to screen several constructs in different expression and purification conditions in parallel. We describe a pipeline, implemented in the Center for Optimized Structural Studies, that enables the systematic screening of expression and purification conditions for recombinant proteins and relies on a series of logical decisions. We first use bioinformatics tools to design a series of protein fragments, which we clone in parallel, and subsequently screen in small scale for optimal expression and purification conditions. Based on a scoring system that assesses soluble expression, we then select the top ranking targets for large-scale purification. In the establishment of our pipeline, emphasis was put on streamlining the processes such that it can be easily but not necessarily automatized. In a typical run of about 2 weeks, we are able to prepare and perform small-scale expression screens for 20–100 different constructs followed by large-scale purification of at least 4–6 proteins. The major advantage of our approach is its flexibility, which allows for easy adoption, either partially or entirely, by any average hypothesis driven laboratory in a manual or robot-assisted manner.     

Single-molecule studies of membrane proteins

Characterizing membrane proteins with single-molecule techniques provides structural and functional insights that cannot be obtained with conventional approaches. Recent studies show that atomic force microscopy (AFM) in the context of a 'lab on a tip' enables the measurement of multiple parameters of membrane proteins. This multifunctional tool can be applied to probe the oligomeric states and conformational changes of membrane protein assemblies in their native environment. The ability to determine diverse properties at high spatial resolution facilitates the mapping of structural flexibilities, electrostatic potentials and electric currents. By using the AFM tip as tweezer, it is possible to characterize unfolding and refolding pathways of single proteins and the location of their molecular interactions. These interactions dictate the stability of the protein and might be modulated by ligands that alter the protein's functional state.     

FTIR spectroscopy of the reaction center of Chloroflexus aurantiacus: photoreduction of the bacteriopheophytin electron acceptor

Mid-infrared spectral changes associated with the photoreduction of the bacteriopheophytin electron acceptor H(A) in reaction centers (RCs) of the filamentous anoxygenic phototrophic bacterium Chloroflexus (Cfl.) aurantiacus are examined by light-induced Fourier transform infrared (FTIR) spectroscopy. The light-induced H(A)(-)/H(A) FTIR (1800-1200cm(-1)) difference spectrum of Cfl. aurantiacus RCs is compared to that of the previously well characterized purple bacterium Rhodobacter (Rba.) sphaeroides RCs. The most notable feature is that the large negative IR band at 1674cm(-1) in Rba. sphaeroides R-26, attributable to the loss of the absorption of the 13(1)-keto carbonyl of H(A) upon the radical anion H(A)(-) formation, exhibits only a very minor upshift to 1675cm(-1) in Cfl. aurantiacus. In contrast, the absorption band of the 131-keto C=O of H(A)(-) is strongly upshifted in the spectrum of Cfl. aurantiacus compared to that of Rba. sphaeroides (from 1588 to 1623cm(-1)). The data are discussed in terms of: (i) replacing the glutamic acid at L104 in Rba. sphaeroides R-26 RCs by a weaker hydrogen bond donor, a glutamine, at the equivalent position L143 in Cfl. aurantiacus RCs; (ii) a strengthening of the hydrogen-bonding interaction of the 131-keto C=O of H(A) with Glu L104 and Gln L143 upon H(A)(-) formation and (iii) a possible influence of the protein dielectric environment on the 131-keto C=O stretching frequency of neutral H(A). A conformational heterogeneity of the 133-ester C=O group of H(A) is detected for Cfl. aurantiacus RCs similar to what has been previously described for purple bacterial RCs.     

Revealing binding sites for myeloperoxidase on the surface of human low density lipoproteins

Low density lipoproteins (LDL) of human blood, once oxidized, provoke cholesterol accumulation in cells of arterial wall, which favors the development of atherosclerosis. Oxidative modification of LDL can result from their interaction with hypochlorous acid produced in the halogenation cycle of myeloperoxidase (MPO). On account that MPO is able to form complexes with LDL it seems important to learn the forces promoting such contacts and to spot the likely binding sites for the enzyme on the surface of LDL particles. In this study affinity chromatography on MPO-Sepharose showed that MPO-LDL complexes are uncoupled at ionic strength above 0.3 M NaCl or when pH of solution goes below 3.6. This is an evidence of ionic interaction between MPO and LDL. We used spin probes of lipid nature embedded in phospholipid monolayer so that a variety of distances between the surface of an LDL particle and the paramagnetic center of a spin probes was provided. Since MPO interaction with labeled LDL caused no alteration of EPR spectra it was concluded that lipid components of LDL are not involved in MPO binding. Analysis of Mn²⁺ distribution between LDL surface and the aqueous milieu showed that the surface negative charge of LDL is not considerably changed upon interaction with MPO. It can be suggested that interaction of LDL with MPO does not involve phospholipids that are the principal carriers of the surface charge. Among synthetic oligopeptides with amino acid sequences mimicking those of apoB-100 fragments – ¹EEEMLEN⁷, ⁵³VELEVPQ⁵⁹ and ⁴⁴⁵EQIQDDCTGDED⁴⁵⁶ – only the latter could replace MPO in the complex with LDL. It is concluded that the likely site of interaction with MPO is the amino acid stretch 445–456 of apoB-100 in LDL.     

Probing the energy landscape of the membrane protein bacteriorhodopsin

The folding and stability of transmembrane proteins is a fundamental and unsolved biological problem. Here, single bacteriorhodopsin molecules were mechanically unfolded from native purple membranes using atomic force microscopy and force spectroscopy. The energy landscape of individual transmembrane alpha helices and polypeptide loops was mapped by monitoring the pulling speed dependence of the unfolding forces and applying Monte Carlo simulations. Single helices formed independently stable units stabilized by a single potential barrier. Mechanical unfolding of the helices was triggered by 3.9-7.7 A extension, while natural unfolding rates were of the order of 10(-3) s(-1). Besides acting as individually stable units, helices associated pairwise, establishing a collective potential barrier. The unfolding pathways of individual proteins reflect distinct pulling speed-dependent unfolding routes in their energy landscapes. These observations support the two-stage model of membrane protein folding in which alpha helices insert into the membrane as stable units and then assemble into the functional protein.