Higher-order epistasis within Pol II trigger loop haplotypes.

RNA polymerase II (Pol II) has a highly conserved domain, the trigger loop (TL), that controls transcription fidelity and speed. We previously probed pairwise genetic interactions between residues within and surrounding the TL for the purpose of understand functional interactions between residues and to understand how individual mutants might alter TL function. We identified widespread incompatibility between TLs of different species when placed in the Saccharomyces cerevisiae Pol II context, indicating species-specific interactions between otherwise highly conserved TLs and its surroundings.

Higher-order epistasis within Pol II trigger loop haplotypes.

RNA polymerase II (Pol II) has a highly conserved domain, the trigger loop (TL), that controls transcription fidelity and speed. We previously probed pairwise genetic interactions between residues within and surrounding the TL for the purpose of understand functional interactions between residues and to understand how individual mutants might alter TL function. We identified widespread incompatibility between TLs of different species when placed in the Pol II context, indicating species-specific interactions between otherwise highly conserved TLs and its surroundings.

Synthesis and characterization of semisynthetic analogs of the antifungal occidiofungin.

Occidiofungin is a broad-spectrum antifungal compound produced by MS14. It is a cyclic glycol-lipopeptide with a novel beta-amino acid (NAA2) containing a hydroxylated C18 fatty acid chain with a xylose sugar. This study reports a strategy to produce semisynthetic analogs of occidiofungin to further explore the structure activity relationships of this class of compounds.

Indole modulates cooperative protein-protein interactions in the flagellar motor.

Indole is a major component of the bacterial exometabolome, and the mechanisms for its wide-ranging effects on bacterial physiology are biomedically significant, although they remain poorly understood. Here, we determined how indole modulates the functions of a widely conserved motility apparatus, the bacterial flagellum. Our experiments in revealed that indole influences the rotation rates and reversals in the flagellum's direction of rotation via multiple mechanisms.

Elucidating the Mechanisms of Action of Antimicrobial Agents.

Despite the ever-growing antibiotic resistance crisis, the rate at which new antimicrobials are being discovered and approved for human use has rapidly declined over the past 75 years. A barrier for advancing newly identified antibiotics beyond discovery is elucidating their mechanism(s) of action. Traditional approaches, such as affinity purification and isolation of resistant mutants, have proven effective but are not always viable options for identifying targets.

Unappreciated Roles for K Channels in Bacterial Physiology.

Potassium (K) channels are highly conserved proteins found in all domains of life, that allow for selective movement of K ions across membranes. Despite their broad distribution, the physiological roles of individual members of this diverse channel family have only been thoroughly explored in eukaryotic systems, where they have critical functions in a variety of cellular processes. Recent studies have demonstrated that bacterial K channels have integral roles in electrical signaling, information propagation, and intercellular communication.

Use of a Fluorescence-Based Assay To Measure Escherichia coli Membrane Potential Changes in High Throughput.

Bacterial membrane potential is difficult to measure using classical electrophysiology techniques due to the small cell size and the presence of the peptidoglycan cell wall. Instead, chemical probes are often used to study membrane potential changes under conditions of interest. Many of these probes are fluorescent molecules that accumulate in a charge-dependent manner, and the resulting fluorescence change can be analyzed via flow cytometry or using a fluorescence microplate reader.

TrkA undergoes a tetramer-to-dimer conversion to open TrkH which enables changes in membrane potential.

TrkH is a bacterial ion channel implicated in K uptake and pH regulation. TrkH assembles with its regulatory protein, TrkA, which closes the channel when bound to ADP and opens it when bound to ATP. However, it is unknown how nucleotides control the gating of TrkH through TrkA.

A Novel Actin Binding Drug with Efficacy.

Occidiofungin is produced by the soil bacterium MS14 and is structurally similar or identical to the burkholdines, xylocandins, and cepacidines. This study identified the primary cellular target of occidiofungin, which was determined to be actin. The modification of occidiofungin with a functional alkyne group enabled affinity purification assays and localization studies in yeast.

High affinity interactions of Pb with synaptotagmin I.

Lead (Pb) is a potent neurotoxin that disrupts synaptic neurotransmission. We report that Synaptotagmin I (SytI), a key regulator of Ca2+-evoked neurotransmitter release, has two high-affinity Pb2+ binding sites that belong to its cytosolic C2A and C2B domains. The crystal structures of Pb2+-complexed C2 domains revealed that protein-bound Pb2+ ions have holodirected coordination geometries and all-oxygen coordination spheres.

Ion Binding to Transport Proteins using Isothermal Titration Calorimetry.

Isothermal titration calorimetry (ITC) is an emerging, label-free technology used to measure ligand binding to membrane proteins. This technology utilizes a titration calorimeter to measure the heat exchange upon ligands binding to proteins, the magnitude of which is based on the overall enthalpy of the reaction. In this protocol, the steps we and others use to measure ion binding to ion transport proteins are described.

A alanine racemase affects spore germination and accommodates serine as a substrate.

has become one of the most common bacterial pathogens in hospital-acquired infections in the United States. Although is strictly anaerobic, it survives in aerobic environments and transmits between hosts via spores. spore germination is triggered in response to certain bile acids and glycine.

Preparation To Minimize Buffer Mismatch in Isothermal Titration Calorimetry Experiments.

There is a growing need to study ligand binding to proteins in native or complex solution using isothermal titration calorimetry (ITC). For example, it is desirable to measure ligand binding to membrane proteins in more native lipid-like environments such as bicelles, where ligands can access both sides of the membrane in a homogeneous environment. A critical step to obtain high signal-to-noise is matching the reaction chamber solution to the ligand solution, typically through a final dialysis or gel filtration step.

Ion-binding properties of a K+ channel selectivity filter in different conformations.

K(+) channels are membrane proteins that selectively conduct K(+) ions across lipid bilayers. Many voltage-gated K(+) (KV) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation.

Determinants of cation transport selectivity: Equilibrium binding and transport kinetics.

The crystal structures of channels and transporters reveal the chemical nature of ion-binding sites and, thereby, constrain mechanistic models for their transport processes. However, these structures, in and of themselves, do not reveal equilibrium selectivity or transport preferences, which can be discerned only from various functional assays. In this Review, I explore the relationship between cation transport protein structures, equilibrium binding measurements, and ion transport selectivity.

Live imaging of inorganic phosphate in plants with cellular and subcellular resolution.

Despite variable and often scarce supplies of inorganic phosphate (Pi) from soils, plants must distribute appropriate amounts of Pi to each cell and subcellular compartment to sustain essential metabolic activities. The ability to monitor Pi dynamics with subcellular resolution in live plants is, therefore, critical for understanding how this essential nutrient is acquired, mobilized, recycled, and stored. Fluorescence indicator protein for inorganic phosphate (FLIPPi) sensors are genetically encoded fluorescence resonance energy transfer-based sensors that have been used to monitor Pi dynamics in cultured animal cells.

Structure of a membrane-embedded prenyltransferase homologous to UBIAD1.

Membrane-embedded prenyltransferases from the UbiA family catalyze the Mg2+-dependent transfer of a hydrophobic polyprenyl chain onto a variety of acceptor molecules and are involved in the synthesis of molecules that mediate electron transport, including Vitamin K and Coenzyme Q. In humans, missense mutations to the protein UbiA prenyltransferase domain-containing 1 (UBIAD1) are responsible for Schnyder crystalline corneal dystrophy, which is a genetic disease that causes blindness. Mechanistic understanding of this family of enzymes has been hampered by a lack of three-dimensional structures.

Equilibrium selectivity alone does not create K+-selective ion conduction in K+ channels.

Potassium (K(+)) channels are selective for K(+) over Na(+) ions during their transport across membranes. We and others have previously shown that tetrameric K(+) channels are primarily occupied by K(+) ions in their selectivity filters under physiological conditions, demonstrating the channel's intrinsic equilibrium preference for K(+) ions. Based on this observation, we hypothesize that the preference for K(+) ions over Na(+) ions in the filter determines its selectivity during ion conduction.

Preferential binding of K+ ions in the selectivity filter at equilibrium explains high selectivity of K+ channels.

K(+) channels exhibit strong selectivity for K(+) ions over Na(+) ions based on electrophysiology experiments that measure ions competing for passage through the channel. During this conduction process, multiple ions interact within the region of the channel called the selectivity filter. Ion selectivity may arise from an equilibrium preference for K(+) ions within the selectivity filter or from a kinetic mechanism whereby Na(+) ions are precluded from entering the selectivity filter.

Traceless protein splicing utilizing evolved split inteins.

Split inteins are parasitic genetic elements frequently found inserted into reading frames of essential proteins. Their association and excision restore host protein function through a protein self-splicing reaction. They have gained an increasingly important role in the chemical modification of proteins to create cyclical, segmentally labeled, and fluorescently tagged proteins.

Structural and thermodynamic properties of selective ion binding in a K+ channel.

Thermodynamic measurements of ion binding to the Streptomyces lividans K(+) channel were carried out using isothermal titration calorimetry, whereas atomic structures of ion-bound and ion-free conformations of the channel were characterized by x-ray crystallography. Here we use these assays to show that the ion radius dependence of selectivity stems from the channel's recognition of ion size (i.e.

Evolutionary information for specifying a protein fold.

Classical studies show that for many proteins, the information required for specifying the tertiary structure is contained in the amino acid sequence. Here, we attempt to define the sequence rules for specifying a protein fold by computationally creating artificial protein sequences using only statistical information encoded in a multiple sequence alignment and no tertiary structure information. Experimental testing of libraries of artificial WW domain sequences shows that a simple statistical energy function capturing coevolution between amino acid residues is necessary and sufficient to specify sequences that fold into native structures.

CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains.

ABC (ATP-binding cassette) proteins constitute a large family of membrane proteins that actively transport a broad range of substrates. Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ABC proteins in that its transmembrane domains comprise an ion channel. Opening and closing of the pore have been linked to ATP binding and hydrolysis at CFTR's two nucleotide-binding domains, NBD1 and NBD2 (see, for example, refs 1, 2).