KMP-11 is a small protein that is believed to control the overall bilayer pressure of the Leishmania parasite. Recent results have suggested that membrane binding and the presence of cholesterol affect the efficacy of Leishmanial infection, in which KMP-11 plays an important role. Nevertheless, there exists no systematic study of membrane interaction with KMP-11 either in the absence or presence of cholesterol. In this article, we investigated the interaction between KMP-11 and phospholipid membranes using an unsaturated (PC 18:1; 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)) and saturated (PC 12:0; 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)) lipid as membrane mimics. Additionally, we studied the effect of cholesterol on the protein-membrane interaction. Steady-state as well as time-resolved fluorescence spectroscopy, isothermal titration calorimetry (ITC), and ζ-potential measurements were used for the determination of the binding constants for the wild-type (WT) and single-site tryptophan mutants. Single-site tryptophan mutants were designed to make sure that the tryptophan residues sample different surface exposures in different mutants. In the absence of cholesterol, the membrane-binding affinities of the partially exposed and buried tryptophan mutants (Y5W and Y48W, respectively) were found to be greater than those of the WT protein. In the presence of cholesterol, the binding constants of the WT and Y48W mutant were found to decrease with an increase in cholesterol concentration. This was in contrast to that in the Y5W and F77W mutants, in which the binding constants increased on adding cholesterol. The present study highlights the interplay among the conformational architecture of a protein, its interaction with the membrane, and membrane composition in modulating the survival of a Leishmania parasite inside host macrophages.

Although the research activities pertaining to the synthesis of fluorescent noble metal nanoclusters (NCs) and their applications in biological optics have been growing, only limited information is available in the near IR (NIR) region. However, fluorescence spectroscopy and microscopy in the NIR region offer significant advantages over UV and visible wavelengths. In this manuscript, we demonstrate biomineralized synthesis of stable Au-Ag bimetallic NCs with tunable NIR fluorescence using bovine serum albumin (BSA) as a protein template. We also demonstrate its application in the detection of toxic heavy metal ions Pb 2+ in vitro and inside cells. The tunability of the fluorescence emission between 680 nm and 815 nm is achieved by systematically varying the ratio of Au and Ag in the composite NCs. The bimetallic NCs when interacting with Pb 2+ offered a large increase in fluorescence intensity, which enabled sensitive detection of Pb 2+ . We determined a limit of detection (LOD) of 96 nM for the detection of Pb 2+ under in vitro conditions, which is significantly less than the safe level in drinking water. Its applicability has also been demonstrated successfully in real water samples collected from local water bodies. † Electronic supplementary information (ESI) available: Two additional tables describing the synthetic optimization in terms of Au/Ag molar ratio variations and time resolved study with respect to Au/Ag variation have been mentioned. Absorption spectra of (Au-Ag@BSA) opt NCs; MALDI mass spectrum of the nanoclusters; X-ray photoelectron spectra; uorescence stability of nanoclusters in the presence of EDTA, NaCl and different pH; stability of (Au-Ag@BSA) opt NCs with time; variation of nanocluster uorescence intensity with increasing Ag concentration. See

Aggregation of Cu-Zn superoxide dismutase (SOD1) is implicated in the motor neuron disease, ALS. Although more than 140 disease mutations of SOD1 are available, their stability or aggregation behaviors in membrane environment are not correlated with disease pathophysiology. Here, we use multiple mutational variants of SOD1 to show that the absence of Zn, and not Cu, significantly impacts membrane attachment of SOD1 through two loop regions facilitating aggregation driven by lipid induced conformational changes. These loop regions influence both the primary (through Cu intake) and the gain of function (through aggregation) of SOD1 presumably through a shared conformational landscape. Combining experimental and theoretical frameworks using representative ALS disease mutants, we develop a 'co-factor derived membrane association model' wherein mutational stress closer to the Zn (but not to the Cu) pocket is responsible for membrane association mediated toxic aggregation and survival time scale after ALS diagnosis.

Virulent Mycobacterium tuberculosis (MTB) strains cause cell death of macrophages (Mϕ) inside TB granuloma using a mechanism which is not well understood. Many bacterial systems utilize toxins to induce host cell damage, which occurs along with immune evasion. These toxins often use chameleon sequences to generate an environmentsensitive conformational switch, facilitating the process of infection. The presence of toxins is not yet known for MTB. Here, we show that MTBsecreted immunogenic MPT63 protein undergoes a switch from β-sheet to helix in response to mutational and environmental stresses. MPT63 in its helical form creates pores in both synthetic and Mϕ membranes, while the native β-sheet protein remains inert toward membrane interactions. Using fluorescence correlation spectroscopy and atomic force microscopy, we show further that the helical form undergoes self-association to produce toxic oligomers of different morphology. Trypan blue and flow cytometry analyses reveal that the helical state can be utilized by MTB for killing Mϕ cells. Collectively, our study emphasizes for the first time a toxin-like behavior of MPT63 induced by an environment-dependent conformational switch, resulting in membrane pore formation by toxic oligomers and Mϕ cell death.

Although significant efforts have been devoted to develop nanoparticle-based biopharmaceuticals, it is not understood how protein conformation and nanoparticle surface modulate each other in optimizing the activity and/or toxicity of the biological molecules. This is particularly important for a protein, which can adopt different conformational states separated by a relatively small energy barrier. In this paper, we have studied nanoparticle binding-induced conformational switch from β-sheet to α-helix of MPT63, a small major secreted protein from Mycobacterium tuberculosis and a drug target against Tuberculosis. The binding of magnetite nanoparticles to MPT63 results in a β-sheet to α-helix switch near the sequence stretch between the 19th and 30th amino acids. As a consequence, the immunogenic response of the protein becomes compromised, which could be restored by protein engineering. This study emphasizes that conformational stability toward NP surface binding may require optimization involving genetic engineering for development of a nanoparticle conjugated pharmaceutical.