Investigating the Chaperoning Effect of Nanoparticles in Chemically Denatured zDHFR: An in vitro Study

Maintenance of native structure and function of the protein is a major concern for industrial production of aggregation prone therapeutically important recombinant proteins. Aggregation may results due to change in the native conformation of proteins under different stress conditions. To overcome the problem of protein aggregation, role of silver and gold nanoparticles have been investigated. The nanoparticles owing to their affirmative interaction with the proteins possess chaperoning activities and protect the native state from denaturation. In the present study, through performing chemical denaturation of zebrafish dihydrofolate reductase using denaturants like guanidine hydrochloride and urea in the presence and absence of gold and silver nanoparticles and monitoring the process through enzyme activity assay and intrinsic tryptophan fluorescence, we have demonstrated the impact of nanoparticles in maintaining native conformation of proteins. Further, the outcome of refolding studies of DHFR protein with nanoparticles monitored by UV-visible spectroscopy was also reported.

Concentration- and pH-Dependent Oligomerization of the Thrombin-Derived C-Terminal Peptide TCP-25

Peptide oligomerization dynamics affects peptide structure, activity, and pharmacodynamic properties. The thrombin C-terminal peptide, TCP-25 (GKYGFYTHVFRLKKWIQKVIDQFGE), is currently in preclinical development for improved wound healing and infection prevention. It exhibits turbidity when formulated at pH 7.4, particularly at concentrations of 0.3 mM or more. We used biochemical and biophysical approaches to explore whether the peptide self-associates and forms oligomers. The peptide showed a dose-dependent increase in turbidity as well as α-helical structure at pH 7.4, a phenomenon not observed at pH 5.0. By analyzing the intrinsic tryptophan fluorescence, we demonstrate that TCP-25 is more stable at high concentrations (0.3 mM) when exposed to high temperatures or a high concentration of denaturant agents, which is compatible with oligomer formation. The denaturation process was reversible above 100 µM of peptide. Dynamic light scattering demonstrated that TCP-25 oligomerization is sensitive to changes in pH, time, and temperature. Computational modeling with an active 18-mer region of TCP-25 showed that the peptide can form pH-dependent higher-order end-to-end oligomers and micelle-like structures, which is in agreement with the experimental data. Thus, TCP-25 exhibits pH- and temperature-dependent dynamic changes involving helical induction and reversible oligomerization, which explains the observed turbidity of the pharmacologically developed formulation.

Chloroplast SRP43 subunit Prevents Aggregation of Proteins

Integration of light-harvesting chlorophyll binding proteins into the thylakoid membrane requires a specific chaperone, being the cpSRP43 subunit, of the signal recognition particle pathway in chloroplasts. cpSRP43, unique to the chloroplast, is responsible for transport of LHCPs through the stroma as well as assisting in the correct folding, assembly and disaggregation of these proteins for the acquisition of light energy. cpSRP43 is a highly flexible, multidomain protein capable of binding distinct partners in the cpSRP pathway. cpSRP43 is an irreplaceable component, necessary for the accurate and successful integration of LHCPs. It can act as a disaggregase without any input of external energy. Its action is based on the ability to associate with variable regions of different proteins owing to the domains and flexibility within its distinctive structure. Understanding the unique capabilities of cpSRP43 in the chloroplast begs the question of its usefulness outside of the plant cell, as well as its yet unknown roles still within the plant cell. Although the capabilities of cpSRP43 as a hub protein, adept to binding many unknown partners, has been alluded to in other works, it has yet to be thoroughly investigated. In this study we discover that cpSRP43 can act as a generic chaperone for proteins other than LHCP/not native to the chloroplast. The high thermal stability of cpSRP43 has been demonstrated in the previous chapter by its ability to retain its secondary structure as well as withstand aggregation upon heating and cooling cycles as confirmed by absorbance, intrinsic tryptophan fluorescence and far UV circular dichroism spectroscopy. This property gives cpSRP43 the basis to act as a generic chaperone and provide protection like that of typical heat shock proteins. Carbonic anhydrase, Concanavalin A and hFGF1 (acidic human fibroblast growth factor), were selected as candidates for chaperoning activity by cpSRP43. In all three cases, heat-induced aggregation of the candidate protein was either eliminated or significantly reduced in the presence of cpSRP43. In the case of hFGF1, the bioactivity was preserved after heat-treatment in the presence of cpSRP43. We have proposed a mechanism by which cpSRP43 is able to execute this action however further investigation is warranted to determine the exact mechanism(s) which may vary dependent on the target protein.

Structural basis for mammalian nucleotide sugar transport

Nucleotide-sugar transporters (NSTs) are critical components of the cellular glycosylation machinery. They transport nucleotide-sugar conjugates into the Golgi lumen, where they are used for the glycosylation of proteins and lipids, and they then subsequently transport the nucleotide monophosphate byproduct back to the cytoplasm. Dysregulation of human NSTs causes several debilitating diseases, and NSTs are virulence factors for many pathogens. Here we present the first crystal structures of a mammalian NST, the mouse CMP-sialic acid transporter (mCST), in complex with its physiological substrates CMP and CMP-sialic acid. Detailed visualization of extensive protein-substrate interactions explains the mechanisms governing substrate selectivity. Further structural analysis of mCST’s unique lumen-facing partially-occluded conformation, coupled with the characterization of substrate-induced quenching of mCST’s intrinsic tryptophan fluorescence, reveals the concerted conformational transitions that occur during substrate transport. These results provide a framework for understanding the effects of disease-causing mutations and the mechanisms of this diverse family of transporters.

Localizing the chaperone activity of erythroid spectrin

ABSTRACTSpectrin, the major protein of the RBC membrane skeleton has canonically been thought to only serve a structural function. We have described a novel chaperone-like property of spectrin and have shown that it is able to prevent the aggregation of other proteins such as alcohol dehydrogenase, insulin and free globin chains. We have tried to localize the molecular origin of chaperone-like activity in multi-domain spectrin by using recombinant spectrin fragments and investigating individual domains. We have characterized the recombinant domains using intrinsic tryptophan fluorescence and CD spectroscopy to show their identity to native spectrin. Hydrophobic ligands Prodan (6-propionyl-2[dimethylamino]-naphthalene) and ANS (1-anilinonaphthalene-8-sulfonic acid) binding has been used to probe the hydrophobicity of the recombinant domains and it is seen that all domains have surface exposed hydrophobic patches; and in accordance with our previous hypothesis only the reconstituted self-association domain binds Prodan. Recombinant domains display comparable chaperone potential in preventing protein aggregation; and substrate selectivity of α-over β-globin is seen. Enzyme refolding studies show alternate pathways of chaperone action. Our current study points to the presence of hydrophobic patches on the surface of these domains as the source of the chaperone activity of spectrin, as notably seen in the self-association domain. There is no one domain largely responsible for the chaperone activity of spectrin; rather all domains appear to contribute equally, such that the chaperone activity of spectrin seems to be a linear sum of the individual activities of the domains.

Proteolysis of β-lactoglobulin by Trypsin: Simulation by Two-Step Model and Experimental Verification by Intrinsic Tryptophan Fluorescence

To distinguish differences in enzymatic hydrolysis of various proteins, we propose an algorithm using a dataset of fluorescence spectra obtained at different moments of hydrolysis t. This algorithm was demonstrated in the example of β-lactoglobulin (β-LG) proteolysis by trypsin. The procedure involved processing the spectra to obtain the wavelength of the maximum fluorescence λmax, which was found to be proportional to the fraction of tryptophanes in hydrated proteolysis products (demasked tryptophanes). Then, the dependence λmax(t) was fitted by biexponential function with two exponential terms, one of which was responsible for the fast part of the fluorescence change during proteolysis. The contribution of this term was quite different for various protein substrates—it was positive for β-LG and negative for β-casein. The observed differences in proteolysis of different substrates were explained by different demasking processes. Combining the fluorescence data with the degrees of hydrolysis of peptide bonds allowed us to analyze the hydrolysis of β-LG in the framework of the two-step proteolysis model and estimate the ratio of rate constants of demasking and hydrolysis and the percentages of initially masked and resistant peptide bonds. This model predicted the existence of a bimodal demasking process with a fast part at the beginning of proteolysis and lag-type kinetics of release for some peptides. Compared with monitoring proteolysis in terms of the degree of hydrolysis only, the fluorescence data are helpful for the recognition of proteolysis patterns.

Effective Concentration of Ionic Liquids for Enhanced Saccharification of Cellulose

In an aqueous enzymatic saccharification using cellulase, the dissolution of crystalline cellulose is one of the rate-limiting steps. Insoluble cellulose powder was preliminarily heat-treated with ionic liquids (ILs), such as [Bmim][Cl] (1-butyl-3-methylimidazolium chloride) and [Amim][Cl] (1-allyl-3-methylimidazolium chloride), which enable the production of soluble cellulose. On the other hand, the presence of ILs leads to a denaturation of enzymes. Using cellulase from Trichoderma viride, the effects of [Bmim][Cl] and [Amim][Cl] in the enzymatic saccharification were compared. The production of glucose was optimized with 5 wt%-ILs, both for [Bmim][Cl] and for [Amim][Cl]. The significant inhibiting effects of ILs (IL concentration >10 wt%) could be due to the denaturation of cellulase, because the peak shifts of intrinsic tryptophan fluorescence were observed in the presence of 7.5 wt%-ILs. To analyze kinetic parameters, the Langmuir adsorption model and the Michaelis-Menten model were employed. The investigation suggests that [Amim][Cl] can provide soluble cellulose more efficiently, and can promote enzymatic saccharification in the IL concentration below 5 wt%.

Effect of cosolvents (polyols) on structural and foaming properties of soy protein isolate

Effect of polyols (mannitol, sorbitol, and xylitol) at three concentrations (5, 10, and 15% w/w) on the structure of soy protein isolates (SPI) was investigated. Changes in foaming properties of SPI were then examined with the addition of polyols at different concentrations. The interactions between SPI and polyols resulted in a substantial decrease in protein surface hydrophobicity and intrinsic tryptophan fluorescence intensity, along with the covering of tyrosine. Furthermore, circular dichroism (CD) spectroscopy of SPI suggested that a more ordered and compact conformation was induced by polyols. Consequently, these structural changes led to lower foamability of SPI. An increase in the viscosity of SPI suspension seemed to be advantageous for improving the foam stability of SPI.