Resumen de Estudio estructural y funcional de proteínas de interés biotecnológico. Aplicaciones y optimización
Víctor Manuel Hernández Rocamora
PaaX is the main regulator protein of the phenylacetic acid (PAA) aerobic degradation pathway in bacteria, acting as a transcriptional repressor in the absence of its inducer phenylacetyl-coenzyme A (PA-CoA). In this Thesis, the overexpression, purification and biochemical characterization of the Escherichia coli W PaaX regulator is reported. This protein formed stable dimers in the presence or absence of PA-CoA. The dissociation constant (Kd) for this ligand was determined by tryptophan fluorescence variation to be 2.4 micromolar. PaaX unfolds non-reversibly at high temperatures, and its solubility and thermostability is highly dependent on ionic strength. On the other hand, PaaX unfolds reversibly upon the addition of the chemical denaturant guanidinium chloride, following a biphasic unfolding curve. At a pH lower that 4, the protein forms an intermediate with a loss of helical structure and the ability to bind the fluorescence probe ANS. In an attempt to increase the protein solubility cysteine to alanine mutations were introduced. None of the four cysteines present in PaaX are absolutely essential for the biological activity of the protein and their change for alanine did not affect PA-CoA binding nor oligomerization.
The C-LytA protein constitutes the choline-binding module of the LytA amidase from Streptococcus pneumoniae. Owing to its affinity for choline and analogs, it is used regularly as an affinity tag for the purification of recombinant proteins. Several salt bridges were engineered on the protein surface in order to create a more thermostable variant. Most effective mutations were pooled to create the C-LytAm7 mutant, which incorporates seven mutations: Y25K, F27K, M33E, N51K, S52K, T85K and T108K. The mutant displayed a 7-8 ºC thermal stabilization compared to the wild-type protein, a complete reversibility upon heating and a higher kinetic stability. Moreover, the mutant accumulates virtually no unfolding intermediates. This mutant can be used as an affinity tag in a wider range of conditions (temperatures up to 90ºC and pHs below 5) where wild-type C-LytA has a much reduced stability.
Choline-binding proteins from Streptococcus pneumoniae recognize specific multivalent choline architectures on the bacterial cell wall, the choline-containing teichoic acids characteristic of this microorganism. As they are involved in essential functions for the virulence of the bacteria as cell division and autolysis, they are attractive targets to fight pneumococcal diseases. Here we report choline-functionalized dendrimeric polymers as potent multivalent inhibitors of choline binding proteins. A 103-104 fold increase in apparent affinity was observed for choline dendrimers compared to free choline both in solution experiments and in surface plasmon resonance experiments. A similar increase in inhibitory potency of four cell wall hydrolases of the CBP family (LytA, LytB, LytC and Pce) was observed. Moreover these compounds were effective in inhibiting autolysis and cell separation in pneumococcal cultures at low micromolar concentrations. These results pave the way for the development of new antimicrobial drugs.
Finally, the previous results allowed us to envisage a generally applicable method for the creation of non-covalent, multivalent complexes of protein with dendrimers. This system uses recombinant proteins fused to the C-LytA affinity tag and choline-derivatized dendrimers as scaffolds. This strategy was tested with the creation of C-LytA-tagged CNA35 and CNA19 fragments of CNA, the collagen-binding adhesin from Staphylococcus aureus. The binding of these fusions to collagen in the presence or absence of choline-derivatized dendrimers was characterized using solid phase binding assays and surface plasmon resonance, demonstrating that the formation of complexes of these proteins with choline-derivatized dendrimers improved the binding properties to collagen. Finally, the complexes were also tested in tissue slices, using fluorescently-labeled choline-derivatized dendrimers, allowing the visualization of collagen fibers in the tissue without labeling the fusion proteins. These results demonstrate the applicability of the system and, in addition, are of direct application in the field of tissue engineering.
