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The periplasmic maltose binding protein, MalE, is a major element in maltose transport and in chemotaxis towards this sugar. Previous genetic analysis of the MalE protein revealed functional domains involved in transport and chemotactic functions. Among them the surface located alpha helix 7, which is part of the C-lobe, one of the two lobes forming the three dimensional structure of MalE. Small deletions in this region abolished maltose transport, although maintaining wild-type affinity and specificity as well as a normal chemoreceptor function. It was suggested that alpha helix 7 may be implicated in interactions between the maltose binding protein and the membrane-bound protein complex (Duplay P, Szmelcman S. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. II. Chemotaxis towards maltose. J Mol Biol 194:675-678: Duplay P, Szmelcman S, Bedouelle H, Hofnung M. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. I: Transport of maltose. J Mol Biol 194:663-673). In this study, we submitted a region of 14 residues--Asp 207 to Gly 220--encompassing alpha helix 7, to genetic analysis by oligonucleotide mediated random mutagenesis. Out of 127 identified mutations, twelve single and five double mutants with normal affinities towards maltose were selected for further investigation. Two types of mutations were characterized, silent mutations that did not affect maltose transport and mutations that heavily impaired transport kinetics, even thought the maltose binding capacity of the mutant proteins remained normal. Three substitutions at Tyr 210 (Y210S, Y210L, Y210N) drastically reduced maltose transport. One substitution at Ala 213 (A213I) and one substitution at Glu 214 (E214K) also impaired transport. These three identified residues, Tyr 210, Ala 213, and Glu 214, which are constituents of alpha helix 7, therefore seem to play some important role in maltose transport, most probably in a productive interaction between the MalE protein and the membrane bound MalFGK2 complex.

The X-ray structures of the maltose bound forms of two insertion/deletion mutants of the Escherichia coli maltodextrin binding protein, MalE322 and MalE178, have been determined and refined. MalE322 involves a one residue deletion, two residue insertion in a hinge segment connecting the two (N and C) domains of the protein, an area already identified as being critical for the correct functioning of the protein. MalE178 involves a nine residue deletion and two residue insertion in a helix at the periphery of the C-domain. The function of both mutant proteins is similar to the wild-type, although MalE322 increases the ability to transport maltose and maltodextrin whilst inhibiting the ability of the cell to grow on dextrins. Both proteins exhibit very localized and conservative conformational changes due to their mutations. The structure of MalE322 shows some deformation of the third hinge strand, indicating the likely cause of change in its biochemistry. MalE178 is stable and its activity virtually unchanged from the wild-type. This is most likely due to the long distance of the mutation from the binding site and conservation of the number of interactions between the area around the deletion site and the main body of the protein.

The periplasmic maltose binding protein, MalE, is a major element in maltose transport and in chemotaxis towards this sugar. Previous genetic analysis of the MalE protein revealed functional domains involved in transport and chemotactic functions. Among them the surface located a helix 7, which is part of the C-lobe, one of the two lobes forming the three dimensional structure of MalE. Small deletions in this region abolished maltose transport, although maintaining wild-type affinity and specificity as well as a normal chemoreceptor function. It was suggested that a helix 7 may be implicated in interactions between the maltose binding protein and the membrane-bound protein complex (Duplay P, Szmelcman S. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. II. Chemotaxis towards maltose. J Mol Biol 794:675-678; Duplay P, Szmelcman S, Bedouelle H, Hofnung M. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. I: Transport of maltose. J Mol Biol 194:663-613). In this study, we submitted a region of 14 residues-Asp 207 to Gly 220-encompassing a helix 7, to genetic analysis by oligonucleotide mediated random mutagenesis. Out of 127 identified mutations, twelve single and five double mutants with normal affinities towards maltose were selected for further investigation. Two types of mutations were characterized, silent mutations that did not affect maltose transport and mutations that heavily impaired transport kinetics, even though the maltose binding capacity of the mutant proteins remained normal. Three substitutions at Tyr 210 (Y210S, Y210L, Y210N) drastically reduced maltose transport. One substitution at Ala 213 (A213I) and one substitution at Glu 214 (E214K) also impaired transport. These three identified residues, Tyr 210, Ala 213, and Glu 214, which are constituents of a helix 7, therefore seem to play some important role in maltose transport, most probably in a productive interaction between the MalE protein and the membrane bound MalFGK2 complex. [ABSTRACT FROM AUTHOR]

The construction of recombinant proteins by genetic engineering has opened new avenues in basic research (studies on protein organization, protein folding, immunogenicity of proteins, ...) and many different applications. Recombinant proteins which keep properties of both parental proteins are especially interesting. For example, if one protein--the vector protein--is targeted to a given cellular compartment, the other protein--the passenger--may be identically targeted. Also, if the vector protein can be purified by a simple affinity chromatographic procedure, this property may be extended to the passenger. The authors have developed a genetic procedure to detect "permissive" sites within potential vector proteins so that genetic fusion to these sites keep most or all biological properties of the vector. When they used LamB, an outer membrane protein from E. coli, foreign sequences could be expressed at the bacterial cell surface. This may lead to several types of applications: live bacterial vaccines, simple diagnostic tests, selection procedures for peptides with biological activity. When they used the MalE protein, a periplasmic maltose binding protein from E. coli, the passengers could be exported and purified in one-step high affinity chromatography in mild non-denaturing conditions. This led us to a simple preparation and purification scheme for the soluble part of the CD4 receptor for the Human Immunodeficiency Virus (HIV).

The periplasmic maltose-binding protein (MBP or MalE protein) of Escherichia coli is an essential element in the transport of maltose and maltodextrins and in the chemotaxis towards these sugars. On the basis of previous results suggesting their possible role in the activity and fluorescence of MBP, we have changed independently to alanine each of the eight tryptophan residues as well as asparagine 294, which is conserved among four periplasmic sugar-binding proteins. Five of the tryptophan mutations affected activity. In four cases (substitution of Trp62, Trp230, Trp232 and Trp340), there was a decrease in MBP affinity towards maltose correlated with modifications in transport and chemotaxis. According to the present state of the 2.3 A three-dimensional structure of MBP, all four residues are in the binding site. Residues Trp62 and Trp340 are in the immediate vicinity of the bound substrate and appear to have direct contacts with maltose; this is in agreement with the drastic increases in Kd values (respectively 67 and 300-fold) upon their substitution by alanine residues. The modest increase in Kd (12-fold) observed upon mutation of Trp230 would be compatible with the lesser degree of interaction this residue has with the bound substrate and the idea that it plays an indirect role, presumably by keeping other residues involved directly in binding in their proper orientation. Substitution of Trp232 resulted in a small increase in Kd value (2-fold) in spite of the fact that this residue is the closest to the ligand of the tryptophan residues according to the three-dimensional model. In the fifth case, replacement of Trp158, which is distant from the binding site, strongly reduced the chemotactic response towards maltose without affecting the transport parameters or the sugar-binding activities of the mutant protein. Trp158 may therefore be specifically implicated in the interaction of MBP with the chemotransducer Tar, but this effect is likely to be indirect, since Trp158 is buried in the structure of MBP. Of course, some structural rearrangements could be responsible in part for the effects of these mutations. The remaining four mutations were silent. The corresponding residues (Trp10, Trp94, Trp129 and Asn294) are all distant from the sugar-binding site on the crystallographic model of MBP, which is in agreement with their lack of effect on binding. In addition, our results show that they play no role in the interactions with the other proteins of the maltose transport (MalF, MalG or MalK) or chemotaxis (Tar) systems.(ABSTRACT TRUNCATED AT 400 WORDS)

The 177 N-terminal amino acids of CD4, the receptor of the human immunodeficiency virus (HIV), have been expressed in Escherichia coli as genetic fusions to the periplasmic maltose-binding protein (MalE) from this organism. A large fraction of the hybrid proteins can be released from the periplasm by osmotic shock and purified in one step on a cross-linked amylose column eluted with maltose under mild conditions. One hybrid protein binds HIV envelope protein gp160 and neutralizes the virus in vitro. This provides the first example of the production and one-step purification of an active form of an eukaryotic protein by fusion to MalE. The use of this system for mass screening of CD4 mutants, high-scale production of the hybrid protein for structural studies on CD4, testing antiviral compounds, and therapeutic assays is discussed.

Athletes need to develop a relatively high muscle mass and low body adipose tissue for the sake of better athletic performance. A full range of nine essential amino acids and eleven non-essential amino acids have to attend in appropriate amounts for protein biosynthesis. The aim of the observational comparative cross-sectional study was to assess the association between the diet quality profile and training-induced muscle mass estimated by bioelectrical impedance among elite male athletes. The research sample comprised 18.1 ± 3.1 year-old Lithuanian professional male athletes (n = 234). The study participants were enrolled to complete 24-h dietary recalls of three non-consecutive days. The body composition was assessed using the bioelectrical impedance analysis (BIA) method. The present study showed a significant insufficiency of the mean carbohydrate intake of 5.7 g/kg/day in a group of aerobic male athletes. The lower muscle mass of aerobic male athletes was related to the lower-carbohydrate diet (adjusted odd ratio (ORadj) 0.3; 95% confidence interval (CI): 0.1–0.7). The mean protein intake of 1.8 g/kg/day was optimal for anabolism in the samples of both anaerobic and aerobic male athletes. The protein intake in appropriate doses was potentially associated with an increase in muscle mass only in anaerobic male athletes (ORadj 2.2; 95% CI: 1.3–3.7). The positive relationship was revealed between the possible muscle mass gain and the increased intakes of amino acids such as isoleucine and histidine among anaerobic athletes (ORadj 2.9; 95% CI: 1.1–4.7 and ORadj 2.9; 95% CI: 1.0–4.3, respectively). An inverse feasible association was indicated between a higher intake of valine and lower muscle mass quantities among anaerobic male athletes (ORadj 0.1; 95% CI: 0.1–0.5). The recommendations for sports nutritionists should emphasize the necessity of advising professional athletes on dietary strategies on how to manipulate dietary amino acid composition with respect to achieving long-term body composition goals. [ABSTRACT FROM AUTHOR]

Several hundred copies of a highly conserved extragenic palindromic sequence, 20-40 nucleotides long, exist along the chromosome of E. coli and S. typhimurium. These have been defined as palindromic units (PU) or repetitive extragenic palindromes (REP). No general function for PUs has been identified. In the present work, we provide data showing that a protein associated with a chromoid extract of E. coli protects PU DNA against exonuclease III digestion. This provides the first experimental evidence that PU constitutes binding sites for a chromoid-associated protein. This result supports the hypothesis that PUs could play a role in the structure of the bacterial chromoid.

Sensory transduction in bacterial chemotaxis is beginning to be understood at the molecular level. At the receptor end, we have some considerable knowledge about the molecular properties of chemoreceptors. At the effector end, we know that flagella rotate and that the direction of rotation is determined by attractants and repellents, although we do not yet know the molecular features of the motor and the gear shift. Between the receptors and the effectors is a system for integrating the sensory transduction, which somewhow involves methylation of membrane proteins and possibly a change in membrane potential, but further details of how the mechanism works remain to be elucidated. It seems to us likely that the facts and concepts learned from a study of sensory transduction in bacteria can be applied to answering questions about transduction mechanisms in eukaryotic cells. Examples include the following: How do sensory stimuli produce their effects in sensory receptor cells? How do neurotransmitters act at receptors of postsynaptic cells to produce the variety of effects possible (changes in membrane potential, in secretion, in contraction, etc.)? How do hormones interact with their receptors to bring about various responses?

The malE gene encodes the periplasmic maltose-binding protein (MBP). Nineteen mutations that still permit synthesis of stable MBP were generated by random insertion of a BamHI octanucleotide into malE and six additional mutations by in-vitro recombinations between mutant genes. The sequence changes were determined; in most cases the linker insertion is accompanied by a small deletion (30 base-pairs on average). The mutant MBP were studied for export, growth on maltose and maltodextrins, maltose transport and binding, and maltose-induced fluorescence changes. Sixteen mutant MBP (out of 21 studied in detail) were found in the periplasmic space: 12 of them retained a high affinity for maltose, and 10 activity for growth on maltose. The results show that several regions of MBP are dispensable for stability, substrate binding and export. Three regions (residues 207 to 220, 297 to 303 and 364 to 370) may be involved in interactions with the MalF or MalG proteins. A region near the C-terminal end is important for maltose binding. Two regions of the mature protein (residues 18 to 42 and 280 to 296) are required for export to, or solubility in, the periplasm.

The kinetic parameters for the maltose transport system in Escherichia coli K12 were determined with maltose and maltotriose as substrates. The system exhibits an apparent Km of 1 muM for maltose and 2 muM for maltotriose. The V of entry was determined as 2.0 and 1.1 nmol substrate/min per 10(8) cells. Mutations in lamB, the structural gene for the receptor protein of phage lambda, increased the Km for maltose transport by a factor of 100-500 without influencing the maximal rate of transport. Maltotriose is no longer transported in these lamB mutants. The maltose-binding protein, an essential component of the maltose transport system, was found to exhibit substrate-dependent fluorescence quenching. This phenomenon was used to determine dissociation constants and to estimate the rate of ligand dissociation. A Kd of 1 muM for maltose and of 0.16 muM for maltotroise was found. From the comparison of the kinetic parameters of transport of maltose and maltotriose in wild-type and lambda-resistant mutants with the binding constants for both sugars to purified maltose-binding protein, we conclude that the lambda receptor facilitates the diffusion of maltose and maltodextrins through the outer membrane.

We examined the chemotactic behavior of ten Escherichia coli mutants able to synthesize a modified periplasmic maltose-binding protein (MBP) retaining high affinity for maltose. Eight were able to grow on maltose (Mal+), two were not (Mal-). In the capillary assay six out of eight of the Mal+ strains showed an optimal response at the same concentration of maltose as the wild-type strain; the amplitude of the response was strongly reduced in two Mal+ mutants and partially affected in one. The amplitude of the chemotactic response of the two Mal- strains was at least equal to that of the wild type, so that the chemotactic and transport functions of MBP were dissociated in these two cases. We define two regions of the protein (residues 297 to 303 and 364 to 369), that are important both for the chemotactic response and for transport, and one region (residues 207 to 220) that is essential for transport but dispensable for chemotaxis. Interestingly, some regions that were found to be inessential for transport are also dispensable for chemotaxis.

Genetic fusions allowing the expression in E. coli of hybrid proteins between a bacterial periplasmic maltose binding protein (MalE) and the CD4 molecule (the receptor of the HIV virus) have been constructed. One of them has kept most of the properties of each constituent: it is exported, can be purified in one step on an affinity column, interacts with anti-MalE and anti-CD4 antibodies, binds HIV gp 120 protein and inactivates HIV virus in an in vitro test.

Periplasmic α-amylase MalS (EC. 3.2.1.1), which belongs to glycoside hydrolase (GH) family 13 subfamily 19, is an integral component of the maltose utilization pathway in Escherichia coli K12 and used among Ecnterobacteriaceae for the effective utilization of maltodextrin. We present the crystal structure of MalS from E. coli and reveal that it has unique structural features of circularly permutated domains and a possible CBM69. The conventional C-domain of amylase consists of amino acids 120–180 (N-terminal) and 646–676 (C-terminal) in MalS, and the whole domain architecture shows the complete circular permutation of C-A-B-A-C in domain order. Regarding substrate interaction, the enzyme has a 6-glucosyl unit pocket binding it to the non-reducing end of the cleavage site. Our study found that residues D385 and F367 play important roles in the preference of MalS for maltohexaose as an initial product. At the active site of MalS, β-CD binds more weakly than the linear substrate, possibly due to the positioning of A402. MalS has two Ca2+ binding sites that contribute significantly to the thermostability of the enzyme. Intriguingly, the study found that MalS exhibits a high binding affinity for polysaccharides such as glycogen and amylopectin. The N domain, of which the electron density map was not observed, was predicted to be CBM69 by AlphaFold2 and might have a binding site for the polysaccharides. Structural analysis of MalS provides new insight into the structure–evolution relationship in GH13 subfamily 19 enzymes and a molecular basis for understanding the details of catalytic function and substrate binding of MalS. [ABSTRACT FROM AUTHOR]

There exists a large body of scientific evidence to support protein intakes in excess of the recommended dietary allowance (RDA) (0.8 g protein/kg/day) to promote the retention of skeletal muscle and loss of adipose tissue during dietary energy restriction. Diet-induced weight loss with as low as possible ratio of skeletal muscle to fat mass loss is a situation we refer to as high-quality weight loss. We propose that high-quality weight loss is often of importance to elite athletes in order to maintain their muscle (engine) and shed unwanted fat mass, potentially improving athletic performance. Current recommendations for protein intakes during weight loss in athletes are set at 1.6-2.4 g protein/kg/day. However, the severity of the caloric deficit and type and intensity of training performed by the athlete will influence at what end of this range athletes choose to be. Other considerations regarding protein intake that may help elite athletes achieve weight loss goals include the quality of protein consumed, and the timing and distribution of protein intake throughout the day. This review highlights the scientific evidence used to support protein recommendations for high-quality weight loss and preservation of performance in athletes. Additionally, the current knowledge surrounding the use of protein supplements, branched chain amino acids (BCAA), β-hydroxy β-methylbutyrate (HMB), and other dietary supplements with weight loss claims will be discussed. [ABSTRACT FROM AUTHOR]

Bacterial defenses against phage, which include CRISPR-mediated immunity and other mechanisms, can carry substantial growth rate costs and can be rapidly lost when pathogens are eliminated. How bacteria preserve their molecular defenses despite their costs, in the face of variable pathogen levels and inter-strain competition, remains a major unsolved problem in evolutionary biology. Here, we present a multilevel model that incorporates biophysics of molecular binding, host-pathogen population dynamics, and ecological dynamics across a large number of independent territories. Using techniques of game theory and non-linear dynamical systems, we show that by maintaining a non-zero failure rate of defenses, hosts sustain sufficient levels of pathogen within an ecology to select against loss of the defense. This resistance switching strategy is evolutionarily stable, and provides a powerful evolutionary mechanism that maintains host-pathogen interactions, selects against cheater strains that avoid the costs of immunity, and enables co-evolutionary dynamics in a wide range of systems. One might think that complete extinction of a virulent pathogen is the most effective way of saving a population. For a bacteria-phage system, Skanata and Kussell show that sustaining a minimum pathogen level is actually favorable to prevent a complete loss of immunity in the long run. [ABSTRACT FROM AUTHOR]

Diarrheal diseases remain a major public health concern worldwide. Many of the causative bacterial pathogens that cause these diseases have a specialized protein complex, the type III secretion system (T3SS), which delivers effector proteins directly into host cells. These effectors manipulate host cell processes for the benefit of the infecting bacteria. The T3SS structure resembles a syringe anchored within the bacterial membrane, projecting toward the host cell membrane. The entry port of the T3SS substrates, called the export apparatus, is formed by five integral membrane proteins. Among the export apparatus proteins, EscV is the largest, and as it forms a nonamer, it constitutes the largest portion of the export apparatus complex. While there are considerable data on the soluble cytoplasmic domain of EscV, our knowledge of its membrane-associated section and its transmembrane domains (TMDs) is still very limited. In this study, using an isolated genetic reporter system, we found that TMD5 and TMD6 of EscV mediate strong self-oligomerization. Substituting these TMDs within the full-length protein with a random hydrophobic sequence resulted in a complete loss of function of the T3SS, further suggesting that the EscV TMD5 and TMD6 sequences have a functional role in addition to their structural role as membrane anchors. As we observed only mild reduction in the ability of the TMD-exchanged variants to integrate into the full or intermediate T3SS complexes, we concluded that EscV TMD5 and TMD6 are not crucial for the global assembly or stability of the T3SS complex but are rather involved in promoting the necessary TMD–TMD interactions within the complex and the overall TMD orientation to allow channel opening for the entry of T3SS substrates. [ABSTRACT FROM AUTHOR]

Maltose binding protein (MBP) is used in recombinant protein expression as an affinity and solubility tag. The monoclonal antibody B48 binds MBP tightly and has no cross‐reactivity to other proteins in an Escherichia coli lysate. This high level of specificity suggested that MBP contains an epitope that could prove useful as a purification and visualization tag for proteins expressed in E. coli. To discover the MBP epitope, a co‐crystal structure was determined for MBP bound to its antibody and four amino acids of MBP were identified as critical for the binding interaction. Fusions of various fragments of MBP to the glutathione S‐transferase protein were engineered in order to identify the smallest fragment still recognized by the α‐MBP antibody. Stabilization of the epitope via mutational engineering resulted in a minimized 14 amino‐acid tag. PDB Code(s): 7JTR; [ABSTRACT FROM AUTHOR]

Successful inter-kingdom relationships are based upon a dynamic balance between defense and cooperation. A certain degree of competition is necessary to guarantee life spread and development. On the other hand, cooperation is a powerful tool to ensure a long lasting adaptation to changing environmental conditions and to support evolution to a higher level of complexity. Bacteria can interact with their (true or potential) parasites (i.e., phages) and with their multicellular hosts. In these model interactions, bacteria learnt how to cope with their inner and outer host, transforming dangerous signals into opportunities and modulating responses in order to achieve an agreement that is beneficial for the overall participants, thus giving rise to a more complex "organism" or ecosystem. In this review, particular attention will be addressed to underline the minimal energy expenditure required for these successful interactions [e.g., moonlighting proteins, post-translational modifications (PTMs), and multitasking signals] and the systemic vision of these processes and ways of life in which the system proves to be more than the sum of the single components. Using an inside-out perspective, I will examine the possibility of multilevel interactions, in which viruses help bacteria to cope with the animal host and bacteria support the human immune system to counteract viral infection in a circular vision. In this sophisticated network, bacteria represent the precious link that insures system stability with relative low energy expenditure. [ABSTRACT FROM AUTHOR]

The MalFGK 2 transporter regulates the movement of maltose across the inner membrane of E. coli and serves as a model system for bacterial ATP binding cassette (ABC) importers. Despite the wealth of biochemical and structural data available, a general model describing the various translocation pathways is still lacking. In this study, we formulate a mathematical model with the goal of determining the transporter reaction pathway, specifically looking at the order of binding events and conformation changes by which transport proceeds. Fitting our mathematical model to equilibrium binding data, we estimate the unknown equilibrium parameters of the system, several of which are key determinants of the transport process. Using these estimates along with steady-state ATPase rate data, we determine which of several possible reaction pathways is dominant, as a function of five underdetermined kinetic parameter values. Because neither experimental measurements nor estimates of certain kinetic rate constants are available, the problem of deciding which of the reaction pathways is responsible for transport remains unsolved. However, using the mathematical framework developed here, a firmer conclusion regarding the dominant reaction pathway as a function of MalE and maltose concentration could be drawn once these unknown kinetic parameters are determined. [ABSTRACT FROM AUTHOR]

Summary: Tomato leaf curl Yunnan virus (TLCYnV)‐encoded C4 protein induces the upregulation of the hypersensitive induced reaction 1 (HIR1) gene but interferes with the HIR1‐mediated hypersensitive response (HR).HIR1 self‐interaction is essential for the HIR1‐induced HR.TLCYnV C4 impairs the HIR1 self‐interaction and concomitantly increases the amount of Leucine‐Rich Repeat protein 1 (LRR1), a modulator of HIR1, which binds to HIR1. LRR1 promotes the degradation of HIR1, compromising the HIR1‐mediated HR.This study provides new insights into the mechanisms employed by a viral protein to counter host resistance through the cooption of the host regulatory system. [ABSTRACT FROM AUTHOR]

The bacterial cytoplasmic membrane separates the cell from its environment and acts as a selective permeability barrier. In addition, it functions in energy conservation, transport, signaling, and biosynthesis processes. Antimicrobial agents disrupting these functions may lead to pleiotropic effects, including leakage of low molecular weight compounds such as ions, amino acids, and ATP and subsequent membrane depolarization. This updated chapter describes two techniques to assess antibiotic-induced membrane impairment in vivo., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The maltose-binding protein (MBP) is one of the most frequently used protein tags due to its capacity to stabilize, solubilize and even crystallize recombinant proteins that are fused to it. Given that MBP is thought to be a highly stable monomeric protein with known characteristics, fused passenger proteins are often studied without being cleaved from MBP. Here we report that a commonly used engineered MBP version (mutated to lower its surface entropy) can form interlaced dimers when fused to short protein sequences derived from the focal adhesion kinase (FAK) or the homologous protein tyrosine kinase 2 (PYK2). These MBP dimers still bind maltose and can interconvert with monomeric forms in vitro under standard conditions despite a contact surface of more than 11,000 Å2. We demonstrate that both the mutations in MBP and the fused protein sequences were required for dimer formation. The FAK and PYK2 sequences are less than 40% identical, monomeric, and did not show specific interactions with MBP, suggesting that a variety of sequences can promote this MBP dimerization. MBP dimerization was abrogated by reverting two of the eight mutations introduced in the engineered MBP. Our results provide an extreme example for induced reversible domain-swapping, with implications for protein folding dynamics. Our observations caution that passenger-promoted MBP dimerization might mislead experimental characterization of the fused protein sequences, but also suggest a simple mutation to stop this phenomenon. [ABSTRACT FROM AUTHOR]

Many gram-negative pathogens utilize a protein complex, termed the type III secretion system (T3SS), to inject virulence factors from their cytoplasm directly into the host cell. An export apparatus that is formed by five putative integral membrane proteins (SctR/S/T/U/V), resides at the center of the T3SS complex. In this study, we characterized the smallest export apparatus protein, SctS, which contains two putative transmembrane domains (PTMD) that dynamically extract from the inner membrane and adopt a helix-turn-helix structure upon assembly of the T3SS. Replacement of each SctS PTMD with an alternative hydrophobic sequence resulted in abolishment of the T3SS activity, yet SctS self- and hetero-interactions as well as the overall assembly of the T3SS complex were unaffected. Our findings suggest that SctS PTMDs are not crucial for the interactions or the assembly of the T3SS base complex but rather that they are involved in adjusting the orientation of the export apparatus relative to additional T3SS sub-structures, such as the cytoplasmic- and the inner-membrane rings. This ensures the fittings between the dynamic and static components of the T3SS and supports the functionality of the T3SS complex. [ABSTRACT FROM AUTHOR]

Cells in all domains of life must translocate newly synthesized proteins both across membranes and into membranes. In eukaryotes, proteins are translocated into the lumen of the ER or the ER membrane. In prokaryotes, proteins are translocated into the cytoplasmic membrane or through the membrane into the periplasm for Gram-negative bacteria or the extracellular space for Gram-positive bacteria. Much of what we know about protein translocation was learned through genetic selections and screens utilizing lacZ gene fusions in Escherichia coli. This review covers the basic principles of protein translocation and how they were discovered and developed. In particular, we discuss how lacZ gene fusions and the phenotypes conferred were exploited to identify the genes involved in protein translocation and provide insights into their mechanisms of action. These approaches, which allowed the elucidation of processes that are conserved throughout the domains of life, illustrate the power of seemingly simple experiments. [ABSTRACT FROM AUTHOR]

The increased prevalence of drug-resistant human pathogenic fungal diseases poses a major threat to global human health. Thus, new drugs are urgently required to combat these infections. Here, we demonstrate that acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acid biosynthesis pathway, is a promising new target for antifungal drug discovery. First, we show that several AHAS inhibitors developed as commercial herbicides are powerful accumulative inhibitors of Candida albicans AHAS (Ki values as low as 800 pM) and have determined high-resolution crystal structures of this enzyme in complex with several of these herbicides. In addition, we have demonstrated that chlorimuron ethyl (CE), a member of the sulfonylurea herbicide family, has potent antifungal activity against five different Candida species and Cryptococcus neoformans (with minimum inhibitory concentration, 50% values as low as 7 nM). Furthermore, in these assays, we have shown CE and itraconazole (a P450 inhibitor) can act synergistically to further improve potency. Finally, we show in Candida albicans-infected mice that CE is highly effective in clearing pathogenic fungal burden in the lungs, liver, and spleen, thus reducing overall mortality rates. Therefore, in view of their low toxicity to human cells, AHAS inhibitors represent a new class of antifungal drug candidates. [ABSTRACT FROM AUTHOR]

In experimental cultures, when bacteria are mixed with lytic (virulent) bacteriophage, bacterial cells resistant to the phage commonly emerge and become the dominant population of bacteria. Following the ascent of resistant mutants, the densities of bacteria in these simple communities become limited by resources rather than the phage. Despite the evolution of resistant hosts, upon which the phage cannot replicate, the lytic phage population is most commonly maintained in an apparently stable state with the resistant bacteria. Several mechanisms have been put forward to account for this result. Here we report the results of population dynamic/evolution experiments with a virulent mutant of phage Lambda, λVIR, and Escherichia coli in serial transfer cultures. We show that, following the ascent of λVIR-resistant bacteria, λVIR is maintained in the majority of cases in maltose-limited minimal media and in all cases in nutrient-rich broth. Using mathematical models and experiments, we show that the dominant mechanism responsible for maintenance of λVIR in these resource-limited populations dominated by resistant E. coli is a high rate of either phenotypic or genetic transition from resistance to susceptibility—a hitherto undemonstrated mechanism we term "leaky resistance." We discuss the implications of leaky resistance to our understanding of the conditions for the maintenance of phage in populations of bacteria—their “existence conditions.” [ABSTRACT FROM AUTHOR]

Catabolite repression is a mechanism that enables bacteria to control carbon utilization. As part of this global regulatory network, components of the phosphoenolpyruvate:carbohydrate phosphotransferase system inhibit the uptake of less favorable sugars when a preferred carbon source such as glucose is available. This process is termed inducer exclusion. In bacteria belonging to the phylum Firmicutes, HPr, phosphorylated at serine 46 (P-Ser46-HPr) is the key player but its mode of action is elusive. To address this question at the level of purified protein components, we have chosen a homolog of the Escherichia coli maltose/maltodextrin ATP-binding cassette transporter from Lactobacillus casei (MalE1-MalF1G1K12) as a model system. We show that the solute binding protein, MalE1, binds linear and cyclic maltodextrins but not maltose. Crystal structures of MalE1 complexed with these sugars provide a clue why maltose is not a substrate. P-Ser46-HPr inhibited MalE1/maltotetraose-stimulated ATPase activity of the transporter incorporated in proteoliposomes. Furthermore, cross-linking experiments revealed that P-Ser46-HPr contacts the nucleotide-binding subunit, MalK1, in proximity to the Walker A motif. However, P-Ser46-HPr did not block binding of ATP to MalK1. Together, our findings provide first biochemical evidence that P-Ser-HPr arrests the transport cycle by preventing ATP hydrolysis at the MalK1 subunits of the transporter. [ABSTRACT FROM AUTHOR]

Although discovered decades ago, the molecular identification, the diversity and versatility of functions, and the evolutionary origin of repeat DNA sequences (REPs) containing palindromic units in prokaryotes are now bringing attention to a wide range of biological scientists. A brief account of the current state of the repeat DNA sequences is presented here. [ABSTRACT FROM AUTHOR]

This study identifies dynamical properties of maltose-binding protein (MBP) useful in unveiling active site residues susceptible to ligand binding. The described methodology has been previously used in support of novel topological techniques of persistent homology and statistical inference in complex, multi-scale, high-dimensional data often encountered in computational biophysics. Here we outline a computational protocol that is based on the anisotropic elastic network models of 14 all-atom three-dimensional protein structures. We introduce the notion of dynamical distance matrices as a measure of correlated interactions among 370 amino acid residues that constitute a single protein. The dynamical distance matrices serve as an input for a persistent homology suite of codes to further distinguish a small subset of residues with high affinity for ligand binding and allosteric activity. In addition, we show that ligand-free closed MBP structures require lower deformation energies than open MBP structures, which may be used in categorization of time-evolving molecular dynamics structures. Analysis of the most probable allosteric coupling pathways between active site residues and the protein exterior is also presented. [ABSTRACT FROM AUTHOR]

This study was conducted to examine the effects of dietary valine on the physical and flavor characteristics, fatty acid (FA) profile, antioxidant status and Nrf2-dependent antioxidant enzyme gene expression in the muscle of young grass carp (Ctenopharyngodon idella) fed increasing levels of valine (4.3, 8.0, 10.6, 13.1, 16.9 and 19.1 g/kg) for 8 weeks. Compared with the control group, the group fed valine showed improved physical characteristics of fish fillets (increased relative shear force, hydroxyproline, protein and lipid levels and decreased cathepsin B and L activities, as well as cooking loss, were observed). Moreover, valine improved the flavor of young grass carp fillets by increasing the amino acid (AA) concentration in fish muscle (increased aspartic acid, threonine, glutamine, cystine, methionine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine and valine concentrations were observed). Additionally, optimal valine supplementation increased the potential health benefits to humans by decreasing the saturated FA (C15:0 and C16:0) concentration and increasing the unsaturated FA (monounsaturated FAs (MUFAs), such as C16:1, C18:1c+t and C20:1, and polyunsaturated FAs (PUFAs), such as C18:3n-3, C20:2 and C22:6) concentration. In addition, the reduced glutathione (GSH) content and the activities of Cu/Zn superoxide dismutase (SOD1), catalase (CAT) and Selenium-dependent glutathione peroxydase (Se-GPx) increased under valine supplementation (P < 0.05). Furthermore, the SOD1, CAT and Se-GPx mRNA levels increased with dietary valine levels, possibly due to the up-regulation of NF-E2-related factor 2 (Nrf2), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1) and the down-regulation of Kelch-like-ECH-associated protein 1 (Keap1) in muscle (P < 0.05). In conclusion, valine improved the physical and flavor characteristics, FA profile, and antioxidant status and regulated the expression of the antioxidant enzyme genes Nrf2, Keap1, TOR and S6K1 in fish fillets. [ABSTRACT FROM AUTHOR]

Bacteria behave differently in space, as indicated by reports of reduced lag phase, higher final cell counts, enhanced biofilm formation, increased virulence, and reduced susceptibility to antibiotics. These phenomena are theorized, at least in part, to result from reduced mass transport in the local extracellular environment, where movement of molecules consumed and excreted by the cell is limited to diffusion in the absence of gravity-dependent convection. However, to date neither empirical nor computational approaches have been able to provide sufficient evidence to confirm this explanation. Molecular genetic analysis findings, conducted as part of a recent spaceflight investigation, support the proposed model. This investigation indicated an overexpression of genes associated with starvation, the search for alternative energy sources, increased metabolism, enhanced acetate production, and other systematic responses to acidity—all of which can be associated with reduced extracellular mass transport. [ABSTRACT FROM AUTHOR]