Sequence and Structural Motifs Controlling the Broad Substrate Specificity of the Mycobacterial Hormone-Sensitive Lipase LipN.

has a complex life cycle transitioning between active and dormant growth states depending on environmental conditions. LipN (Rv2970c) is a conserved mycobacterial serine hydrolase with regulated catalytic activity at the interface between active and dormant growth conditions. LipN also catalyzes the xenobiotic degradation of a tertiary ester substrate and contains multiple conserved motifs connected with the ability to catalyze the hydrolysis of difficult tertiary ester substrates.

Structure-guided microbial targeting of antistaphylococcal prodrugs.

Carboxy ester prodrugs are widely employed to increase oral absorption and potency of phosphonate antibiotics. Prodrugging can mask problematic chemical features that prevent cellular uptake and may enable tissue-specific compound delivery. However, many carboxy ester promoieties are rapidly hydrolyzed by serum esterases, limiting their therapeutic potential.

Transition metal cation inhibition of Mycobacterium tuberculosis esterase RV0045C.

Mycobacterium tuberculosis virulence is highly metal-dependent with metal availability modulating the shift from the dormant to active states of M. tuberculosis infection. Rv0045c from M.

Fluorogenic structure activity library pinpoints molecular variations in substrate specificity of structurally homologous esterases.

Cellular esterases catalyze many essential biological functions by performing hydrolysis reactions on diverse substrates. The promiscuity of esterases complicates assignment of their substrate preferences and biological functions. To identify universal factors controlling esterase substrate recognition, we designed a 32-member structure-activity relationship (SAR) library of fluorogenic ester substrates and used this library to systematically interrogate esterase preference for chain length, branching patterns, and polarity to differentiate common classes of esterase substrates.

Measuring the Global Substrate Specificity of Mycobacterial Serine Hydrolases Using a Library of Fluorogenic Ester Substrates.

Among the proteins required for lipid metabolism in Mycobacterium tuberculosis are a significant number of uncharacterized serine hydrolases, especially lipases and esterases. Using a streamlined synthetic method, a library of immolative fluorogenic ester substrates was expanded to better represent the natural lipidomic diversity of Mycobacterium. This expanded fluorogenic library was then used to rapidly characterize the global structure activity relationship (SAR) of mycobacterial serine hydrolases in M.

Implementation of a Collaborative Series of Classroom-Based Undergraduate Research Experiences Spanning Chemical Biology, Biochemistry, and Neurobiology.

Classroom undergraduate research experiences (CUREs) provide students access to the measurable benefits of undergraduate research experiences (UREs). Herein, we describe the implementation and assessment of a novel model for cohesive CUREs focused on central research themes involving faculty research collaboration across departments. Specifically, we implemented three collaborative CUREs spanning chemical biology, biochemistry, and neurobiology that incorporated faculty members' research interests and revolved around the central theme of visualizing biological processes like Mycobacterium tuberculosis enzyme activity and neural signaling using fluorescent molecules.

Distinct substrate selectivity of a metabolic hydrolase from Mycobacterium tuberculosis.

The transition between dormant and active Mycobacterium tuberculosis infection requires reorganization of its lipid metabolism and activation of a battery of serine hydrolase enzymes. Among these serine hydrolases, Rv0045c is a mycobacterial-specific serine hydrolase with limited sequence homology outside mycobacteria but structural homology to divergent bacterial hydrolase families. Herein, we determined the global substrate specificity of Rv0045c against a library of fluorogenic hydrolase substrates, constructed a combined experimental and computational model for its binding pocket, and performed comprehensive substitutional analysis to develop a structural map of its binding pocket.

Investigations on the Q and CT Bands of Cytochrome c Submonolayer Adsorbed on an Alumina Surface Using Broadband Spectroscopy with Single-Mode Integrated Optical Waveguides.

In this work, we report experimental results on the molar absorptivity of cytochrome c adsorbed at different submonolayer levels onto an aluminum oxide waveguide surface; our data show a clear dependence of the protein optical properties on its surface density. The measurements were performed using the broadband, single-mode, integrated optical waveguide spectroscopic technique, which is an extremely sensitive tool able to reach submonolayer levels of detection required for this type of studies. This investigation focuses on the molar absorptivity at the Q-band (centered at 525 nm) and, for the first time to our knowledge, the weak charge transfer (CT) band (centered at 695 nm) of surface-adsorbed cyt c.

Stabilization of yeast cytochrome C covalently immobilized on fused silica surfaces.

The surface-enhanced conformational stability of yeast cytochrome c (YCC) covalently immobilized on a fused silica prism with heterobifunctional cross-linkers has been studied by attenuated total reflection absorption spectroscopy using the Soret band of the heme prosthetic group as a probe. Comparison of the results to those of horse cytochrome c physisorbed on the same substrate as well as to the corresponding proteins in solution indicates that the surface plays a significant role in stabilizing the native conformation of the surface-bound YCC. Unfolding to extended configurations was so hindered that the native conformation of the covalently immobilized protein is essentially unaffected by the presence of denaturants such as methanol and 1-propanol.

DNA polymerase template interactions probed by degenerate isosteric nucleobase analogs.

The development of novel artificial nucleobases and detailed X-ray crystal structures for primer/template/DNA polymerase complexes provide opportunities to assess DNA-protein interactions that dictate specificity. Recent results have shown that base pair shape recognition in the context of DNA polymerase must be considered a significant component. The isosteric azole carboxamide nucleobases (compounds 1-5; ) differ only in the number and placement of nitrogen atoms within a common shape and therefore present unique electronic distributions that are shown to dictate the selectivity of template-directed nucleotide incorporation by DNA polymerases.