Development Of New Probes And Inhibitors For Protein Fibrillations And Their Application In Photocatalytic Regeneration Of Nicotinamide Cofactors

Protein fibrillation is associated with a diversity of human neurodegenerative diseases,such as Parkinson's syndrome,type ? diabetes,Huntington's disease and Alzheimer's disease.Therefore,the research of amyloid protein has attracted extensive attention in the field of biomedical science.It is very important regarding diagnostic and therapeutic potential to develop new molecules which can monitor the dynamics of protein fibrillation and inhibit the formation of protein fibrils.Furthermore,amyloid proteins contain unique high stability and other physical and chemical properties due to its unique periodic cross-?-folded conformation.Therefore,it is also critical to expand the application of amyloid protein-based functional materials.Based on two kinds of amyloid proteins,we explored their capability in photocatalytic coenzyme regeneration.The major contributions of this study are as follows:1.Crocein Orange G mediated detection and modulation of amyloid fibrillation revealed by surface-enhanced Raman spectroscopyHerein,we identified and characterized a novel inhibitor called Crocein Orange G(COG),which has the ability that inhibiting the nucleation of insulin and impeding the protofibril formation,revealed by various experimental approaches as well as molecular docking.In order to modulate the process,there are a number of fibrillation inhibitors have been reported,although their working mechanism remains vague,calling for new means to decipher their interaction.In particular,the surface-enhanced Raman spectroscopy(SERS)helps to identify the binding sites and illustrate the interaction mechanism between the COG and insulin.Moreover,the label-free detection of fibrillation process was achieved using SERS and the AgIMNPs served as enhancement substrate.Combining with molecular docking and SERS,the SERS-based approach provides structural information concerning protein-ligand interaction and protein fibrillation.This study demonstrated that the SERS can become a new powerful means to study the interaction between inhibitors and amyloid proteins,furthermore,and it can potentially be a common tool for amyloid research.Strikingly,the SERS signal of COG corresponds very well with the state of protein fibrillation,hinting its function as an amyloid SERS signal amplifier.Therefore,this study provides a new means to monitor and interfere amyloid fibrillation2.Live cell fluorescent stain of bacterial curli and biofilm through supramolecular recognition between bromophenol blue and CsgACsgA is an extracellular frizzled protein fibril produced by bacteria as a structural component of biofilms,which has recently emerged as a platform for manufacturing living materials through integrating distinct functional components into curli on the cell surfaces of the biofilms.The identification of curli-specific dyes for biofilm communities of microorganisms is of great value.We describe here the development of a curli fluorescent light-up probe called bromophenol blue(BPB),which can specifically bind to curli within the biofilm in situ via recognizing CsgA,the main building-block of curli.A linear response for CsgA fibril detection in the 1.5-40 ?? concentration range was visualized,with the limit of detection 0.7 ??.In the presence of 1 % serum,CsgA fibrils can be reliably detected with recoveries 98.6 %-104.2 % and relative standard deviations of less than 3.16 %.We expect this platform provide a new perspective for the research on biofilm,amyloid disease and living materials.3.Bromophenol blue combine with self-assembling functional amyloid protein CsgA to form supramolecular living complexes for the photocatalytic regeneration of NADHBased on the work in the previous chapter,we explored the application of BPB and amyloid CsgA co-assembly system and their application in NADH photocatalytic regeneration.As a special type of functional amyloid,CsgA has a highly ordered nanostructure with high stability,insolubility and robustness.We investigate the photocatalytic energy transfer from chromophores to catalytic units in a manner similar to natural optical systems,utilizing the functional amyloid protein as a scaffold to hold and arrange BPB in high density on its surface.We successfully coupled the light collection module for NADH regeneration(light reaction)with the enzyme reduction reaction module(dark reaction),demonstrating that the BPB-amyloid hybridized nanostructure is suitable for the chemical conversion from solar energy to biocatalysis.4.PGHMs as a Photocatalyst for Light-Driven Regeneration of Nicotinamide Cofactors and Solar-to-Chemical ConversionPhotoenzyme coupling catalysis,though integrating inorganic materials and enzyme catalysis,shows great potential in light-driven synthesis.The application of protein capped gold nanoclusters(AuNCs)in photocatalysis has not yet been explored.Herein,we report a series of highly fluorescent self-assembled protein-gold hybrid materials(PGHMs)with three different emission wavelengths are fabricated to enable the highly efficient visible light-driven photocatalytic reduction of nicotinamide adenine dinucleotide(NAD+).The photocatalytic performance and kinetic processes are investigated to evaluate the efficiency of NADH regeneration based on the PGHM platform.The photocatalytic performances of AuNCs of PGHM-B have a higher solarto-chemical conversion efficiency than others under visible light irradiation.Furthermore,bio-abiotic hybrid photosynthetic systems containing PGHMs–oxidoreductase/non-photosynthetic bacteria are constructed to efficiently produce glutathione(GSH)and potentially other biochemical products.The present research highlights the development of new inorganic biohybrid systems for the efficient synthetic biological solar energy conversion and utilization.