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Arginine has been used extensively as an excipient in the formulation development of protein- based biopharmaceuticals. We investigate the role of arginine in suppressing protein aggregation and its mechanism by using bovine serum albumin as a model system. By using sedimentation velocity and other analytical techniques, we show that the use of arginine inhibits temperature-induced aggregation of the protein. We use fluorescence correlation spectroscopy and other spectroscopic techniques to show that arginine inhibits accumulation of partially folded intermediates, potentially involved in the aggregation process. The hydrodynamic radii of the protein in its native, unfolded, and intermediate states have been determined using fluorescence correlation spectroscopy at single-molecule resolution. A possible mechanism of the effects of arginine and its role as an aggregation suppressor has been discussed. [ABSTRACT FROM AUTHOR]

Binding interactions of KMP-11 with the IL choline dihydrogen phosphate (CDHP) solvent have been explored from multispectroscopic studies in conjunction with docking and molecular dynamics simulations. The protein KMP-11, a vaccine candidate against 'Leishmaniasis', does not contain any tryptophan (Trp) in its WT sequence. For this study, two single site Trp mutant proteins Y5W and Y89W of KMP -11 have been generated as Trp used as biomarker by the aid of intrinsic fluorescence. In the former mutant (Y5W) Trp is inserted in the vicinity of the N-terminal, while in the later (Y89W), the same residue is disposed near the C-terminal of the protein. The binding interactions of CDHP with both the mutant proteins have been explored from UV–Vis, steady state and time resolved fluorescence spectroscopic studies. The bimolecular quenching rate constant (k q) at various temperatures, as obtained from fluorescence spectroscopic studies suggest definite but weak binding interactions between the mutant proteins and CDHP IL solvent. This result is further corroborated from the near and far - UV Circular Dichroism (CD) spectra. The possible binding sites of CDHP in the vicinity of WT and mutant proteins and the corresponding binding forces have been predicted from the molecular docking studies. The results of MD simulation further portend minimal or no unfolding in the secondary structures of the WT and the mutant proteins upon addition of CDHP molecule. We believe that this in-depth study featuring the interaction of KMP-11 with IL CDHP will enlighten the fundamental understanding towards the development of improved protein based vaccines in general and KMP-11 guided vaccines in particular for the prevention of VL. Unlabelled Image • Binding interactions of KMP-11 and its Trp mutants with ionic liquid (IL) CDHP • Interactions explored by both multi-spectroscopic and theoretical studies. • Quenching of fluorescence and the thermodynamic parameters were estimated. • Changes in protein conformation suggested by near and far CD measurements. • Enlightening the development of protein based vaccine against visceral Leishmaniasis [ABSTRACT FROM AUTHOR]

Genetic switches frequently include DNA loops secured by proteins. Recent studies of the lambda bacteriophage repressor (CI), showed that this arrangement in which the protein links two sets of three operators separated by approximately 2.3 kbp, optimizes both the stability and dynamics of DNA loops, compared to an arrangement with just two sets of two operators. Because adjacent dimers interact pairwise, we hypothesized that the odd number of operators in each set of the lambda regulatory system might have evolved to allow for semi-specific, pair-wise interactions that add stability to the loop while maintaining it dynamic. More generally, additional CI dimers may bind non-specifically to flanking DNA sequences making the genetic switch more sensitive to CI concentration. Here, we tested this hypothesis using spectroscopic and imaging approaches to study the binding of the lambda repressor (CI) dimer protein to DNA fragments. For fragments with only one operator and a short flanking sequence, fluorescence correlation spectroscopy measurements clearly indicated the presence of two distinct DNA-CI complexes; one is thought to have a non-specifically bound CI dimer on the flanking sequence. Scanning force micrographs of CI bound to DNA with all six operators revealed wild-type or mutant proteins bound at operator positions. The number of bound, wild-type proteins increased with CI concentration and was larger than expected for strictly specific binding to operators. In contrast, a mutant that fails to oligomerize beyond a dimer, D197G, only bound to operators. These data are evidence that CI cooperativity promotes oligomerization that extends from operator sites to influence the thermodynamics and kinetics of CI-mediated looping. [ABSTRACT FROM AUTHOR]

The aggregation of α-synuclein(A-syn) has been implicatedin the pathogenesis of Parkinson’s disease (PD). Although theearly events of aggregation and not the matured amyloid fibrils arebelieved to be responsible for the toxicity, it has been difficultto probe the formation of early oligomers experimentally. We studiedthe effect of Fe3O4nanoparticle (NP) in theearly stage of aggregation of A-syn using fluorescence correlationspectroscopy (FCS) and laser scanning microscopy. The binding betweenthe monomeric protein and NPs was also studied using FCS at single-moleculeresolution. Our data showed that the addition of bare Fe3O4NPs accelerated the rate of early aggregation, andit did not bind the monomeric A-syn. In contrast, l-lysine(Lys)-coated Fe3O4NPs showed strong bindingwith the monomeric A-syn, inhibiting the early events of aggregation.Lys-coated Fe3O4NPs showed significantly lesscell toxicity compared with bare Fe3O4NPs andcan be explored as a possible strategy to develop therapeutic applicationagainst PD. To the best of our knowledge, this report is the firstexample of using a small molecule to attenuate the early (and arguablythe most relevant in terms of PD pathogenesis) events of A-syn aggregation. [ABSTRACT FROM AUTHOR]