John Cavanagh

Sensitivity improvement in proton-detected two-dimensional heteronuclear correlation NMR spectroscopy

Abstract The sensitivity of proton-detected two-dimensional heteronuclear correlation NMR spectroscopy can be increased by as much as a factor of √2 relative to that of the conventional methods, for heteronuclei with a single attached proton. The enhanced sensitivity is obtained by refocusing and detecting two orthogonal in-phase proton magnetization components, rather than the single component recorded conventionally. The two magnetization components are deconvoluted to produce two pure-phase spectra that are added together to produce a spectrum with an enhanced signal-to-noise ratio. Methods for improving the sensitivity of the main classes of heteronuclear correlation experiments are presented and the effect of relaxation during the new experiments on the sensitivity enhancement is discussed. The new methods are demonstrated by comparing conventional and sensitivity-enhanced 1H-15N heteronuclear correlation spectra of bovine pancreatic trypsin inhibitor at 15N natural abundance.

Millisecond-timescale motions contribute to the function of the bacterial response regulator protein Spo0F

Protein backbones and side chains display varying degrees of flexibility, which allows many slightly different but related conformational substates to occur. Such fluctuations are known to differ in both timescale and magnitude, from rotation of methyl groups (nanoseconds) to the flipping of buried tyrosine rings (seconds),. Because many mechanisms for protein function require conformational change, it has been proposed that some of these ground-state fluctuations are related to protein function. But exactly which aspects of motion are functionally relevant remains to be determined. Only a few examples so far exist where function can be correlated to structural fluctuations with known magnitude and timescale,. As part of an investigation of the mechanism of action of the Bacillus subtilis response regulator Spo0F, we have explored the relationship between the motional characteristics and protein–protein interactions. Here we use a set of nuclear magnetic resonance 15N relaxation measurements to determine the relative timescales of Spo0F backbone fluctuations on the picosecond-to-millisecond timescale. We show that regions having motion on the millisecond timescale correlate with residues and surfaces that are known to be critical for protein–protein interactions.

Synergistic Effects between Conventional Antibiotics and 2-Aminoimidazole-Derived Antibiofilm Agents

ABSTRACT 2-Aminoimidazoles are an emerging class of small molecules that possess the ability to inhibit and disperse biofilms across bacterial order, class, and phylum. Herein, we report the synergistic effect between a 2-aminoimidazole/triazole conjugate and antibiotics toward dispersing preestablished biofilms, culminating with a 3-orders-of-magnitude increase of biofilm dispersion toward Staphylococcus aureus biofilms. Furthermore, we document that the 2-aminoimidazole/triazole conjugate will also resensitize multidrug-resistant strains of bacteria to the effects of conventional antibiotics, including methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Acinetobacter baumannii.

Sensitivity improvement in isotropic mixing (TOCSY) experiments

Abstract A simple modification of the two-dimensional TOCSY experiment which leads to a √2 improvement in sensitivity is described. This improvement is achieved by retaining the magnetization transfer pathways from both of the orthogonal, in-phase magnetization components which are present during the isotropic mixing period. In addition, a simple baseline correction procedure which can greatly improve the quality of NMR spectra is described.

Neuropathy Target Esterase Is Required for Adult Vertebrate Axon Maintenance

The enzyme neuropathy target esterase (NTE) is present in neurons and deacylates the major membrane phospholipid, phosphatidylcholine (PtdCho). Mutation of the NTE gene or poisoning by neuropathic organophosphates—chemical inhibitors of NTE—causes distal degeneration of long spinal axons in humans. However, analogous neuropathological changes have not been reported in nestin-cre:NTEfl/fl mice with NTE-deficient neural tissue. Furthermore, altered PtdCho homeostasis has not been detected in NTE-deficient vertebrates. Here, we describe distal degeneration of the longest spinal axons in ∼3-week-old nestin-cre:NTEfl/fl mice and in adult C57BL/6J mice after acute dosing with a neuropathic organophosphate: in both groups early degenerative lesions were followed by swellings comprising accumulated axoplasmic material. In mice dosed acutely with organophosphate, maximal numbers of lesions, in the longest spinal sensory axon tract, were attained within days and were preceded by a transient rise in neural PtdCho. In nestin-cre:NTEfl/fl mice, sustained elevation of PtdCho over many months was accompanied by progressive degeneration and massive swelling of axons in sensory and motor spinal tracts and by increasing hindlimb dysfunction. Axonal lesion distribution closely resembled that in hereditary spastic paraplegia (HSP). The importance of defective membrane trafficking in HSP and the association of NTE with the endoplasmic reticulum—the starting point for the constitutive secretory pathway and transport of neuronal materials into axons—prompted investigation for a role of NTE in secretion. Cultured NTE-deficient neurons displayed modestly impaired secretion, consistent with neuronal viability and damage in vivo initially restricted to distal parts of the longest axons.

Abh and AbrB Control of Bacillus subtilis Antimicrobial Gene Expression

The Bacillus subtilis abh gene encodes a protein whose N-terminal domain has 74% identity to the DNA-binding domain of the global regulatory protein AbrB. Strains with a mutation in abh showed alterations in the production of antimicrobial compounds directed against some other Bacillus species and gram-positive microbes. Relative to its wild-type parental strain, the abh mutant was found deficient, enhanced, or unaffected for the production of antimicrobial activity. Using lacZ fusions, we examined the effects of abh upon the expression of 10 promoters known to be regulated by AbrB, including five that transcribe well-characterized antimicrobial functions (SdpC, SkfA, TasA, sublancin, and subtilosin). For an otherwise wild-type background, the results show that Abh plays a negative regulatory role in the expression of four of the promoters, a positive role for the expression of three, and no apparent regulatory role in the expression of the other three promoters. Binding of AbrB and Abh to the promoter regions was examined using DNase I footprinting, and the results revealed significant differences. The transcription of abh is not autoregulated, but it is subject to a degree of AbrB-afforded negative regulation. The results indicate that Abh is part of the complex interconnected regulatory system that controls gene expression during the transition from active growth to stationary phase.

Structure, binding interface and hydrophobic transitions of Ca(2+)-loaded calbindin-D(28K).

Calbindin-D28K is a Ca2+-binding protein, performing roles as both a calcium buffer and calcium sensor. The NMR solution structure of Ca2+-loaded calbindin-D28K reveals a single, globular fold consisting of six distinct EF-hand subdomains, which coordinate Ca2+ in loops on EF1, EF3, EF4 and EF5. Target peptides from Ran-binding protein M and myo-inositol monophosphatase, along with a new target from procaspase-3, are shown to interact with the protein on a surface comprised of α5 (EF3), α8 (EF4) and the EF2-EF3 and EF4-EF5 loops. Fluorescence experiments reveal that calbindin-D28K adopts discrete hydrophobic states as it binds Ca2+. The structure, binding interface and hydrophobic characteristics of Ca2+-loaded calbindin-D28K provide the first detailed insights into how this essential protein may function. This structure is one of the largest high-resolution NMR structures and the largest monomeric EF-hand protein to be solved to date.

Anti-Biofilm Compounds Derived from Marine Sponges

Bacterial biofilms are surface-attached communities of microorganisms that are protected by an extracellular matrix of biomolecules. In the biofilm state, bacteria are significantly more resistant to external assault, including attack by antibiotics. In their native environment, bacterial biofilms underpin costly biofouling that wreaks havoc on shipping, utilities, and offshore industry. Within a host environment, they are insensitive to antiseptics and basic host immune responses. It is estimated that up to 80% of all microbial infections are biofilm-based. Biofilm infections of indwelling medical devices are of particular concern, since once the device is colonized, infection is almost impossible to eliminate. Given the prominence of biofilms in infectious diseases, there is a notable effort towards developing small, synthetically available molecules that will modulate bacterial biofilm development and maintenance. Here, we highlight the development of small molecules that inhibit and/or disperse bacterial biofilms specifically through non-microbicidal mechanisms. Importantly, we discuss several sets of compounds derived from marine sponges that we are developing in our labs to address the persistent biofilm problem. We will discuss: discovery/synthesis of natural products and their analogues—including our marine sponge-derived compounds and initial adjuvant activity and toxicological screening of our novel anti-biofilm compounds.

THE TIME-DEPENDENCE OF COHERENCE TRANSFER IN HOMONUCLEAR ISOTROPIC MIXING EXPERIMENTS

Abstract The fundamental behavior of coherence transfer in isotropic mixing experiments (TOCSY or HOHAHA) is examined. A numerical simulation program has been written to calculate the time dependence of coherence transfer under the isotropic scalar coupling interaction for arbitrary spin systems. Results are shown for a variety of amino acids and for deoxyribose, which should facilitate the selection of optimum mixing times in practical applications of the TOCSY experiment.

Amide Isosteres of Oroidin: Assessment of Antibiofilm Activity and C. elegans Toxicity

The synthesis and antibiofilm activities of sulfonamide, urea, and thiourea oroidin analogues are described. The most active derivative was able to selectively inhibit P. aeruginosa biofilm development and is also shown to be nontoxic upward of 1 mM to the development of C. elegans in comparison to other similar isosteric analogues and the natural product oroidin.