Mitochondrial Acyl Carrier Protein of Leishmania major Displays Features Distinct from the Canonical Type II ACP.

Prokaryotes synthesize fatty acids using a type II synthesis pathway (FAS). In this process, the central player, i.e., the acyl carrier protein (ACP), sequesters the growing acyl chain in its internal hydrophobic cavity. As the acyl chain length increases, the cavity expands in size, which is reflected in the NMR chemical shift perturbations and crystal structures of the acyl-ACP intermediates. A few eukaryotic organelles, such as plastids and mitochondria, also harbor type II fatty acid synthesis machinery. Plastid FAS from spinach and Plasmodium falciparum has been characterized at the molecular level, but the mitochondrial pathway remains unexplored. Here, we report NMR studies of the mitochondrial acyl-acyl carrier protein intermediates of Leishmania major (acyl-LmACP). Our studies show that LmACP experiences remarkably small conformational changes upon acylation, with perturbations confined to helices II and III only. CastP determined that the cavity size of apo-LmACP (PDB entry 5ZWT) is less than that of Escherichia coli ACP (PDB 1T8K). Thus, the small chemical shift perturbations observed in the LmACP intermediates, coupled with CastP results, suggest an unusually small cavity when fully expanded. The faster rate of C8-LmACP chain hydrolysis compared to E. coli ACP (EcACP) also supports these convictions. Structure comparison of LmACP with other type II ACP disclosed unique differences in the helix I and loop I conformations, as well as several residues present there. Numerous hydrophobic residues in helix I and loop I (conserved in all mitochondrial ACPs) are substituted with hydrophilic residues in the bacterial/plastid type II ACP. For instance, Phe and leucine at positions 14 and 34 in LmACP are substituted with a hydrophilic residue and Ala in bacterial/plastid type II ACP. Mutation of Leu 34 to Ala (corresponding residue in EcACP) resulted in a complete loss of structure, underscoring its importance in maintaining the ACP fold. Thus, our NMR studies, combined with insights from the crystal structure, highlight several unique features of LmACP, distinct from the prokaryote and plastid type II ACP. Given the high sequence identity, the features might be conserved in all mitochondrial ACPs.

Lipoate protein ligase B primarily recognizes the C8-phosphopantetheine arm of its donor substrate and weakly binds the acyl carrier protein.

Lipoic acid is a sulfur-containing cofactor indispensable for the function of several metabolic enzymes. In microorganisms, lipoic acid can be salvaged from the surroundings by lipoate protein ligase A (LplA), an ATP-dependent enzyme. Alternatively, it can be synthesized by the sequential actions of lipoate protein ligase B (LipB) and lipoyl synthase (LipA). LipB takes up the octanoyl chain from C8-acyl carrier protein (C8-ACP), a byproduct of the type II fatty acid synthesis pathway, and transfers it to a conserved lysine of the lipoyl domain of a dehydrogenase. However, the molecular basis of its substrate recognition is still not fully understood. Using Escherichia coli LipB as a model enzyme, we show here that the octanoyl-transferase mainly recognizes the 4'-phosphopantetheine-tethered acyl-chain of its donor substrate and weakly binds the apo-acyl carrier protein. We demonstrate LipB can accept octanoate from its own ACP and noncognate ACPs, as well as C8-CoA. Furthermore, our 1H saturation transfer difference and 31P NMR studies demonstrate the binding of adenosine, as well as the phosphopantetheine arm of CoA to LipB, akin to binding to LplA. Finally, we show a conserved 71RGG73 loop, analogous to the lipoate-binding loop of LplA, is required for full LipB activity. Collectively, our studies highlight commonalities between LipB and LplA in their mechanism of substrate recognition. This knowledge could be of significance in the treatment of mitochondrial fatty acid synthesis related disorders.

A GX2GX3G motif facilitates acyl chain sequestration by Saccharomyces cerevisiae acyl carrier protein.

Saccharomyces cerevisiae acyl carrier protein (ScACP) is a component of the large fungal fatty acid synthase I (FAS I) complex. ScACP comprises two subdomains: a conserved ACP domain that shares extensive structural homology with other ACPs and a unique structural domain. Unlike the metazoan type I ACP that does not sequester the acyl chain, ScACP can partially sequester the growing acyl chain within its hydrophobic core by a mechanism that remains elusive. Our studies on the acyl-ScACP intermediates disclose a unique 188GX2GX3G195 sequence in helix II important for ACP function. Complete loss of sequestration was observed upon mutation of the three glycines in this sequence to valine (G188V/G191V/G195V), while G191V and G188V/G191V double mutants displayed a faster rate of acyl chain hydrolysis. Likewise, mutation of Thr216 to Ala altered the size of the hydrophobic cavity, resulting in loss of C12- chain sequestration. Combining NMR studies with insights from the crystal structure, we show that three glycines in helix II and a threonine in helix IV favor conformational change, which in turn generate space for acyl chain sequestration. Furthermore, we identified the primary hydrophobic cavity of ScACP, present between the carboxyl end of helix II and IV. The opening of the cavity lies between the second and third turns of helix II and loop II. Overall, the study highlights a novel role of the GX2GX3G motif in regulating acyl chain sequestration, vital for ScACP function.

Role of nuclear localization signals in the DNA delivery function of Chikungunya virus capsid protein.

Capsids of several RNA viruses are reported to have unconventional roles attributed to their subcellular trafficking property. The capsid of CHIKV is also found to localize in the nucleus, but the rationale is not yet clear. To understand the role of the nuclear-localized capsid, we examined the nucleic acid binding and cargo delivery activity of the CHIKV capsid. We used bacterially purified capsid protein to probe the binding affinity with CHIKV genome-specific and non-specific nucleic acids. We found that the capsid was able to bind non-specifically to different forms of nucleic acids. The successful transfection of GFP-tagged plasmid DNA by CHIKV capsid protein shows the DNA delivery ability of the protein. Further, we selected and investigated the DNA binding and cargo delivery activity of commercially synthesized Nuclear Localization Signal sequences (NLS 1 and NLS2) of capsid protein. Both peptides showed comparable DNA binding affinity, however, only the NLS1 peptide was capable of delivering plasmid DNA inside the cell. Furthermore, the cellular uptake study using the FITC-labelled NLS1 peptide was performed to highlight the membrane penetrating ability. Structural analysis was performed using circular dichroism and NMR spectroscopy to elucidate the transfection ability of the NLS1 peptides. Our findings suggest that the capsid of CHIKV might influence cellular trafficking in the infected cell via non-specific interactions. Our study also indicates the significance of NLS sequences in the multifunctionality of CHIKV capsid protein.

Differential expression of structural and functional proteins during bean common mosaic virus-host plant interaction.

Bean common mosaic virus (BCMV), the most common seed-borne pathogen in Phaseolus vulgaris L. is known to cause severe loss in productivity across the globe. In the present study, proteomic analyses were performed for leaf samples from control (healthy) and susceptible BCMV infected plants. The differential expression of proteins was evaluated using two-dimensional gel electrophoresis (2-DE). Approximately, 1098 proteins were spotted, amongst which 107 proteins were observed to be statistically significant with differential expression. The functional categorization of the differential proteins illustrated that they were involved in biotic/abiotic stress (18%), energy and carbon metabolism (11%), photosynthesis (46%), protein biosynthesis (10%), chaperoning (5%), chlorophyll (5%) and polyunsaturated fatty acid biosynthesis (5%). This is the first report on the comparative proteome study of compatible plant-BCMV interactions in P. vulgaris which contributes largely to the understanding of protein-mediated disease resistance/susceptible mechanisms.

The "Recognition Helix" of the Type II Acyl Carrier Protein (ACP) Utilizes a "Ubiquitin Interacting Motif (UIM)"-like Surface To Bind Its Partners.

Interaction interfaces comprise a finite number of sequence/structural motifs, which are often repeated in nature. Here we show how a helical motif present in the acyl carrier protein (ACP), involved in multiprotein interactions, displays a binding interface similar to that of the "ubiquitin interacting motif (UIM)". Analysis of the crystal structures of the ACP-enzyme complexes gave the first hint that helix II of the ACP ("universal recognition helix") utilizes UIM-like noncovalent interactions to associate with the type II fatty acid biosynthesis (FAS) pathway enzymes. The ACP interacting functional surface of the FAS enzymes comprises positively charged residues, flanking a central hydrophobic patch, akin to ubiquitin. Our nuclear magnetic resonance chemical shift perturbation studies, relaxation studies, and surface plasmon resonance measurements unequivocally show that helix II of ACP behaves like a UIM motif in its interactions with ubiquitin, by binding the Ile 44 functional surface of the latter. A synthetic peptide with the ACP helix II sequence showed equivalent binding to ubiquitin. The evolution of similar interaction motifs in the two systems has probably been driven by functional constraints, as both ACP and UIM participate in multiprotein interactions.

The Structure of the Holo-Acyl Carrier Protein of Leishmania major Displays a Remarkably Different Phosphopantetheinyl Transferase Binding Interface.

The genome of Leishmania major encodes a type II fatty acid biosynthesis pathway for which no structural or biochemical information exists. Here, for the first time, we have characterized the central player of the pathway, the acyl carrier protein (LmACP), using nuclear magnetic resonance (NMR). Structurally, the LmACP molecule is similar to other type II ACPs, comprising a four-helix bundle, enclosing a hydrophobic core. Dissimilarities in sequence, however, exist in helix II (recognition helix) of the protein. The enzymatic conversion of apo-LmACP into the holo form using type I (Escherichia coli AcpS) and type II (Sfp type) phosphopantetheinyl transferases (PPTs) is relatively slow. Mutagenesis studies underscore the importance of the residues present at the protein-protein interaction interface of LmACP in modulating the activity of PPTs. Interestingly, the cognate PPT for this ACP, the L. major 4'-phosphopantetheinyl transferase (LmPPT), does not show any enzymatic activity toward it, though it readily converts other type I and type II ACPs into their holo forms. NMR chemical shift perturbation studies suggest a moderately tight complex between LmACP and its cognate PPT, suggesting inhibition. We surmise that the unique surface of LmACP might have evolved to complement its cognate enzyme (LmPPT), possibly for the purpose of regulation.

The apo-acyl coenzyme A binding protein of Leishmania major forms a unique 'AXXA' motif mediated dimer.

Acyl-Coenzyme A binding domain containing proteins (ACBDs) are ubiquitous in nearly all eukaryotes. They can exist as a free protein, or a domain of a large, multidomain, multifunctional protein. Besides modularity, ACBDs also display multiplicity. The same organism may have multiple ACBDs, differing in sequence and organization. By virtue of this diversity, ACBDs perform functions ranging from transport, synthesis, trafficking, signal transduction, transcription, and gene regulation. In plants and some microorganisms, these ACBDs are designated ACBPs (acyl-CoA binding proteins). The simplest ACBD/ACBP is a small, ∼10 kDa, soluble protein, comprising the acyl-CoA binding (ACB) domain. Most of these small ACBDs exist as monomers, while a few show a tendency to oligomerize. In sync with those studies, we report the crystal structure of two ACBDs from Leishmania major, named ACBP103, and ACBP96 based on the number of residues present. Interestingly, ACBP103 crystallized as a monomer and a dimer under different crystallization conditions. Careful examination of the dimer disclosed an exposed 'AXXA' motif in the helix I of the two ACBP103 monomers, aligned in a head-to-tail arrangement in the dimer. Glutaraldehyde cross-linking studies confirm that apo-ACBP103 can self-associate in solution. Isothermal titration calorimetry studies further show that ACBP103 can bind ligands ranging from C8 - to C20-CoA, and the data could be best fit to a 'two sets of sites'/sequential binding site model. Taken together, our studies show that Leishmania major ACBP103 can self-associate in the apo-form through a unique dimerization motif, an interaction that may play an important role in its function.

Exploring Anticancer Properties of Medicinal Plants against Breast Cancer by Downregulating Human Epidermal Growth Factor Receptor 2.

Plants have a history of being employed in managing breast cancer. However, no scientific evidence supports the idea that these plants can effectively reduce the level of HER2 expression. In this study, extracts from 10 medicinal plants were evaluated for their anticancer properties against HER2-positive breast cancer cells through various methods, including the SRB assay, comet assay, annexin V-FITC dual staining, and immunoblotting. All extracts exerted antiproliferative activity against HER2-positive breast cancer cells. Furthermore, Terminalia chebula (T. chebula), Berberis aristata (B. aristata), and Mucuna pruriens (M. pruriens) reduced HER2 expression in tested cell lines. In addition, an increased Bax/Bcl-2 ratio was observed after the treatment. A comparative proteomics study showed modulation in the proteome profile of breast cancer cells after treatment with T. chebula, B. aristata, Punica granatum, M. pruriens, and Acorus calamus. Metabolic profiling of lead plants revealed the existence of multiple anticancer compounds. Our study demonstrates the considerable potential of the mentioned plants as innovative therapies for HER2-positive breast cancer.

Fatty acid synthesis suppresses dietary polyunsaturated fatty acid use.

Dietary polyunsaturated fatty acids (PUFA) are increasingly recognized for their health benefits, whereas a high production of endogenous fatty acids - a process called de novo lipogenesis (DNL) - is closely linked to metabolic diseases. Determinants of PUFA incorporation into complex lipids are insufficiently understood and may influence the onset and progression of metabolic diseases. Here we show that fatty acid synthase (FASN), the key enzyme of DNL, critically determines the use of dietary PUFA in mice and humans. Moreover, the combination of FASN inhibition and PUFA-supplementation decreases liver triacylglycerols (TAG) in mice fed with high-fat diet. Mechanistically, FASN inhibition causes higher PUFA uptake via the lysophosphatidylcholine transporter MFSD2A, and a diacylglycerol O-acyltransferase 2 (DGAT2)-dependent incorporation of PUFA into TAG. Overall, the outcome of PUFA supplementation may depend on the degree of endogenous DNL and combining PUFA supplementation and FASN inhibition might be a promising approach to target metabolic disease.

Therapeutic Potential of Low-Intensity Magnetic Field Stimulation in 6-Hydroxydopamine Rat Model of Parkinson's Disease: From Inflammation to Motor Function.

Background: Parkinson's disease (PD) is a progressive neurodegenerative disorder that mainly affects the aged population. Transcranial magnetic field (MF) stimulation has shown to provide temporary motor recovery in neurological disorders. Purpose: The aim of this study was to understand the cellular and molecular mechanism of low-intensity MF stimulation (17.96 µT; 50Hz; 2 h/day, four weeks) in a rat model of severe PD. Methods: A clinically relevant, bilateral striatal 6-hydroxydopamine (6-OHDA) lesioned rat model of severe PD was employed to test the efficacy of low-intensity MF stimulation in the management of motor symptoms. The mechanism of action of MF was dissected by assessing the microglial activation, tissue ultrastructure, and cerebrospinal fluid (CSF) metabolomics using microdialysis. Results: We observed a significant improvement in the postural balance and gait after MF exposure with a significant reduction in the number of activated microglia. There was an improvement in striatal dopaminergic innervation and glutamate levels but it did not reach a level of statistical significance. Conclusion: MF stimulation helped ameliorate the motor deficits and reduced inflammation but was unable to provide a significant change in terms of dopaminergic innervation and metabolic profile in the severe 6-OHDA PD rat model.

STAT3 inhibition mediated upregulation of multiple immune response pathways in dengue infection.

Dengue infection is a world-wide public health threat infecting millions of people annually. Till date no specific antiviral or vaccine is available against dengue virus. Recent evidence indicates that targeting host STAT3 could prove to be an effective antiviral therapy against dengue infection. To explore the potential of STAT3 inhibition as an antiviral strategy, we utilized a STAT3 inhibitor stattic as antiviral agent and performed whole proteome analysis of mammalian cells by mass spectrometry. Differentially expressed proteins among the infected and stattic treated groups were sorted based on their fold change expression and their functional annotation studies were carried out to establish their biological networks. The results presented in the current study indicated that treatment with stattic induces several antiviral pathways to counteract dengue infection. Together with this, we also observed that treatment with stattic downregulates pathways involved in viral transcription and translation thus establishing STAT3 as a suitable target for the development of antiviral interventions. This study establishes the role of STAT3 inhibition as an alternative strategy to counteract DENV pathogenesis. Targeting STAT3 by stattic or similar molecules may help in identifying novel therapeutic interventions against DENV and probably other flaviviruses.

Conformational and dynamic changes at the interface contribute to ligand binding by ubiquitin.

Ubiquitin interacts with numerous domains and motifs in its lifetime that vary in structure but bind the same hydrophobic patch. To identify the structural features of ubiquitin that make it an exceptional protein-protein interaction partner, we have studied the interaction of ubiquitin with the signal transducing adaptor molecule-1 ubiquitin interacting motif (UIM) using nuclear magnetic resonance. Our studies bring to light the role of the inherent backbone flexibility of ubiquitin in its interactions with a large array of binding partners, revealed from the changes in C(α) chemical shifts, backbone dynamics, and hydrogen bond lengths upon UIM binding. The crystal structures of ubiquitin complexes lend further support to our findings, underscoring the importance of the unique and flexible hydrogen bond network within ubiquitin and simultaneously providing insights into the nature of the slow motions. Taken together, our studies provide an in-depth view of the molecular changes associated with ligand recognition by ubiquitin.

Structural and mechanistic insights into modulation of α-Synuclein fibril formation by aloin and emodin.

α-Synuclein (α-Syn) aggregation/fibrillation is a leading cause of neuronal death and is one of the major pathogenic factors involved in the progression of Parkinson's' disease (PD). Against this backdrop, discovering new molecules as inhibitors or modulators of α-Syn aggregation/fibrillation is a subject of enormous research. In this study, we have shown modulation, disaggregation, and neuroprotective potential of aloin and emodin against α-Syn aggregation/fibrillation. Thioflavin T (ThT) fluorescence assay showed an increase in lag phase from (51.14 ± 2) h to (68.58 ± 2) h and (74.14 ± 3) h in the presence of aloin and emodin respectively. ANS binding assay represents a modulatory effect of these molecules on hydrophobicity which is crucial for aggregates/fibril formation. NMR spectroscopy and tyrosine quenching studies reveal the binding of aloin/emodin with monomeric α-Syn. TEM and DLS micrographs illustrate the attenuating effect of aloin/emodin against the development of large aggregates/fibrils. Our seeding experiments suggest aloin/emodin generate seeding incompetent oligomers that direct the off-pathway aggregation/fibrillation. Also, aloin/emodin capably reduces the fibrils-induced cytotoxicity and disassembles the preexisting amyloid fibrils. These findings provide deep insight into the modulatory mechanism of α-Syn aggregation/fibrillation in the presence of aloin and emodin, thereby suggesting their potential roles as promising therapeutic molecules against aggregation/fibrillation related disorders.

Heme-iron ligand (M80-Fe) in cytochrome c is destabilizing: combined in vitro and in silico approaches to monitor changes in structure, stability and dynamics of the protein on mutation.

Structure, stability and dynamics properties of horse cytochrome c (cyt c) and its genetically engineered M80G mutant have been investigated. The nature of the Met80 axial ligation to heme iron is believed to be the major determinant of the oxidation-reduction reactions inside and outside the cell of a particular cytochrome. This ligation has played an important role in the studies of protein structure, stability and protein folding/unfolding. To understand this ligation better, Met80 of horse cyt c has been mutated to Gly that is unable to bind to the heme iron. We have examined the effect of the M80G mutation on the structure and stability of the WT (wild type) protein by using absorbance spectroscopy, far-UV, near-UV and Soret circular dichroism, fluorescence spectroscopy and differential scanning calorimetry. We have observed that mutation caused a partial loss of secondary and tertiary structure with slightly increased overall stability of the protein. We have also measured the dynamic behavior of WT cyt c and its M80G mutant in the oxidized form (Fe3+) using the essential dynamics (ED) method. A 400 ns MD simulations were run for WT cyt c and its mutant M80G in water using GROMOS96 force field. MD results revealed that the stability and flexibility increased in mutant M80G (Fe…S (Met80) bond removed). Essential dynamics analysis revealed that the first five eigenvectors were mainly involved in overall motions of WT cyt c and its M80G mutant but the amplitude of concerted motions decreased in M80G mutant relative to WT cyt c.Communicated by Ramaswamy H. Sarma.

Chemical shift assignments of the biotin carboxyl carrier protein domain of L. major Methylcrotonyl-CoA carboxylase.

Methylcrotonyl-CoA carboxylase (MCCC) is a biotin dependent enzyme, that plays a crucial role in leucine metabolism. The enzyme comprises a biotin carboxylase (BC), a carboxyltransferase (CT), and a biotin carboxyl carrier protein (BCCP) domain. MCCC is synthesized as an apo-protein, and is posttranslationally modified at a lysine residue, conserved in the biotin carboxyl carrier protein (BCCP) domain. In order to understand the structure, function and interactions of L. major MCCC, we have expressed and characterized its domains. Here we report the complete chemical shift assignments of MCCC BCCP domain of L. major. Furthermore, we have used the assignments to generate a model of the same, using CS-Rosetta. We have also followed its chemical shift perturbations upon biotin modification. Changes were observed at the lysine 51 amide, that undergoes biotin modification, and a few others present in its immediate neighborhood.

An overview of the fatty acid biosynthesis in the protozoan parasite Leishmania and its relevance as a drug target against leishmaniasis.

Leishmaniasis is one of the fast-growing parasitic diseases worldwide. The treatment of this fatal disease presents a daunting challenge because of its adverse effects, necessity for long-term treatment regime, unavailability of functional drugs, emergence of drug resistance and the related expenditure. This calls for an urgent need for novel drugs and the evaluation of new targets. Proteins of the fatty acid biosynthetic pathway are validated as drug targets in pathogenic bacteria and certain viruses. Likewise, this pathway has been speculated as a suitable target against parasite infections. Fatty acid synthesis in parasites seems to be very complex and distinct from the counterpart mammalian host due to the presence of unique mechanisms for fatty acid biosynthesis and acquisition. In recent times, there have been few evidences of the existence of this pathway in the bloodstream form of some pathogens. The fatty acid biosynthesis thus presents a viable and attractive target for emerging therapeutics. Understanding the mechanisms underlying fatty acid metabolism is key to identifying a potential drug target. However, investigations in this direction are still limited and this article attempts to outline the existing knowledge, while highlighting the scope and relevance of the fatty acid biosynthetic pathway as a drug target. This review highlights the advances in the treatment of leishmaniasis, the importance of lipids in the pathogen, known facts about the fatty acid biosynthesis in Leishmania and how this pathway can be manipulated to combat leishmaniasis, suggesting novel drug targets.

Ellagic Acid Modulates Uninduced as well as Mutation and Metal-Induced Aggregation of α-Synuclein: Implications for Parkinson's Disease.

α-Synuclein (αS) is an intrinsically disordered protein whose aggregation and deposition in Lewy bodies is involved in the progression of Parkinson's disease (PD) and other related disorders. The aggregation process of αS is also triggered by mutations like A53T and E46K in the SNCA gene and disruption in metal-ion homeostasis. Currently, there is no obviating therapy available in the market that could effectively prevent the progression of the disease. In this backdrop, there exists an emerging need to consider naturally occurring polyphenols and flavonoids as potential therapeutic agents against PD. In this study, we demonstrate the modulatory effect of ellagic acid (EA) against wild-type as well as mutation and metal-induced aggregation of αS. Thioflavin T (ThT) fluorescence assay suggests that EA acts on the nucleation phase of αS fibrillization, thereby increasing the lag phase from 21.33 ± 3.01 to 48.20 ± 5.05 h and reducing the fibrils growth rate from 4.60 ± 2.06 to 0.890 ± 0.36 h-1. 8-Anilino-1-naphthalene sulfonic acid (ANS), Congo red (CR), and intrinsic fluorescence studies indicate that the interaction of EA with αS facilitates the structural changes in the protein that lead to inhibition of fibril formation. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) images illustrate that the size of fibrils diminishes up to 100 nm in the presence of EA. Dot blot and seeding experiments put forward that EA directs the αS aggregation toward off-pathway fibrillization. Our 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay deciphers the role of EA in minimizing the αS fibril-induced toxicity, thereupon leading to an increase in cell viability. Also, EA attenuates both mutations as well as metal-induced αS fibrillization and disaggregates the preexisting fibrils. Additionally, computational studies elucidate that EA preferentially interacts with the N-terminal and NAC domain of αS. Hence, this work reveals the aggregation inhibition mechanism of EA and provides considerable therapeutic interventions against PD and related disorders.

L. major apo-acyl carrier protein forms ordered aggregates due to an exposed phenylalanine, while phosphopantetheine inhibits aggregation in the holo-form.

L. major acyl carrier protein (ACP) is a mitochondrial protein, involved in fatty acid biosynthesis. The protein is expressed as an apo-protein, and post-translationally modified at Ser 37 by a 4'-Phosphopantetheinyl transferase. Crystal structure of the apo-form of the protein at pH 5.5 suggests a four helix bundle fold, typical of ACP's. However, upon lowering the pH to 5.0, it undergoes a conformational transition from α-helix to ß-sheet, and displays amyloid like properties. When left for a few days at room temperature at this pH, the protein forms fibrils, visible under Transmission electron microscopy (TEM). Using an approach combining NMR, biophysical techniques, and mutagenesis, we have identified a Phe residue present on helix II of ACP, liable for this change. Phosphopantetheinylation of LmACP, or mutation of Phe 45 to the corresponding residue in E. coli ACP (methionine), slows down the conformational change. Conversely, substitution of methionine 44 of E. coli ACP with a phenylalanine, causes enhanced ThT binding. Thus, we demonstrate the unique property of an exposed Phe in inducing, and phophopantetheine in inhibiting amyloidogenesis. Taken together, our study adds L. major acyl carrier protein to the list of ACPs that act as pH sensors.

Stability of uniformly labeled (13C and 15N) cytochrome c and its L94G mutant.

Cytochrome c (cyt c) is widely used as a model protein to study (i) folding and stability aspects of the protein folding problem and (ii) structure-function relationship from the evolutionary point of view. Databases of cyts c now contain 285 cyt c sequences from different organisms. A sequence alignment of all these proteins with respect to horse cyt c led to several important conclusions. One of them is that Leu94 is always conserved in all 30 mammalian cyts c. It is known that mutation L94G of the wild type (WT) horse cyt c is destabilizing and mutant exists as molten globule under the native condition (buffer pH 6 and 25 °C). We have expressed and purified uniformly labeled (13C and 15N) and unlabeled WT horse cyt c and its L94G mutant. We report that labeling does not affect the thermodynamic stability of proteins. To support this conclusion, the secondary and tertiary structure of each protein in labeled and unlabeled forms was determined by conventional techniques (UV-Vis absorption and circular dichroism spectroscopy).