Browsing by Subject "Molecular chemistry"

Gram-negative bacteria possess an asymmetric outer membrane in which the inner leaflet is composed primarily of phospholipids while the outer leaflet contains both phospholipids and lipopolysaccharide (LPS). LPS forms a structural barrier that protects Gram-negative bacteria from antibiotics and other environmental stressors. The lipid A anchor of LPS is a glucosamine-based saccharolipid that is further modified with core and O-antigen sugars. In addition to serving a structural role as the hydrophobic anchor of LPS, lipid A is recognized by the innate immune system in animal cells and macrophages. The enzymes of Lipid A biosynthesis are conserved in Gram-negative bacteria; in most species, a single copy of each bio-synthetic gene is present. The exception is lpxH, which is an essential gene encoding a membrane-associated UDP-2,3-diacylglucosamine hydrolase, which catalyzed the attack of water upon the alpha-phosphate of its substrate and the leaving of UMP, resulting in the formation of lipid X. Many Gram-negatives lack an lpxH orthologue, yet these species must possess an activity analogous to that of LpxH. We used bioinformatics approaches to identify a candidate gene, designated lpxI, encoding this activity in the model organism Caulobacter crescentus. We then demonstrated that lpxI can rescue Escherichia coli deficient in lpxH. Moreover, we have shown that LpxI possesses robust and specific UDP-2,3-diacylglucosamine hydrolase activity in vitro. We have developed high-yield purification schema for Caulobacter crescentus LpxI (CcLpxI) heterologously expressed in E. coli. We crystallized CcLpxI and determined its 2.6 Å x-ray crystal structure in complex with lipid X. CcLpxI, which has no known homologues, consists of two novel domains connected by a linker. Moreover, we have identified a point mutant of CcLpxI which co-purifies with its substrate in a 0.85:1 molar ratio. We have solved the x-ray crystal structure of this mutant to 3.0 Å; preliminary comparison with the product-complexed model reveals striking differences. The findings described herein set the stage for further mechanistic and structural characterization of this novel enzyme.

In this work, we also isolate and characterize LpxB, an essential lipid A biosynthetic gene which is conserved among all Gram-negative bacteria. We purify E. coli and Hemophilus influeznea LpxB to near-homogeneity on a 10 mg scale, and we determine that E. coli LpxB activity is dependent upon the bulk surface concentration of its substrates in a mixed micellar assay system, suggesting that catalysis occurs at the lipid interface. E. coli LpxB partitions with membranes, but this interaction is partially abolished in high-salt conditions, suggesting that a significant component of LpxB's membrane association is ionic in nature. E. coli LpxB (Mr ~ 43 kDa) is a peripheral membrane protein, and we demonstrate that it co-purifies with phospholipids. We estimate, by autoradiography and mass-spectrometry, molar ratios of phospholipids to purified enzyme of 1.6-3.5:1. Transmission electron microscopy reveals the accumulation of intra-cellular membranes when LpxB is massively over-expressed. Alanine-scanning mutagenesis of selected conserved LpxB residues identified two, D89A and R201A, for which no residual catalytic activity is detected. Our data support the hypothesis that LpxB performs catalysis at the cytoplasmic surface of the inner membrane, and provide a rational starting-point for structural studies. This work contributes to knowledge of the small but growing set of structurally and mechanistically characterized enzymes which perform chemistry upon lipids.

Library display technologies have enabled the development of peptides with affinity for a given substrate. Such affinity-capture reagents have driven progress in many fields, from basic biochemistry to neuropharmacology. A major limitation in the development of neuro-pharmaceuticals has been an inability to examine how the behavioral effects of drugs are mediated by each of the distinct yet intermingled cell types in any given brain region. DART (Drugs Acutely Restricted by Tethering) is the first method to overcome this technical barrier, enabling the delivery of therapeutics to a precise genetically defined neuronal cell type. At the core of DART’s specificity is a capture of a chemical Rx-HTL (HaloTag Ligand conjugated to a drug) by a genetically encoded HTP (HaloTag protein), creating an artificial dosing window. Although the technology has already revealed novel neurobiological insights, a narrow dosing window currently limits DART to neurobiological questions where dose can be tightly controlled, such as via intracranial infusion over a small brain volume. Our goal is to adapt the principles of directed evolution and library display to improve the dosing window of DART and enable its brain-wide delivery. Moreover, using the same principles, we aim to develop an orthogonal DART pair for multiplexed delivery of any combination of drugs to two distinct cell types. The underlying principle of a library display tool is a physical linkage between phenotype (a protein) and genotype (its corresponding nucleotide sequence). This conjugated mRNA, encoding the displayed protein, serves as a unique identifier for each variant. Over the past three decades, several display systems have been developed, each with a unique set of limitations. Typically, there is a tradeoff between the stability of this linkage and the number of unique variants (library size). Thus, no existing platform offers the desired trifecta of linkage stability, library size, and product yield. This work introduces a novel in vitro protein display technology called GRIP Display (Gluing RNA to Its Protein) that permits the generation and simultaneous screening of vast protein libraries (~10^14 variants) against a target of interest, with minimal genetic cross-talk, significant selection enrichment, and one-step simple experimental protocol. Here, we demonstrate 1) the development of GRIP Display and its utility in the optimization of a large binding tunnel of HTP to enhance the covalent capture of its chemical ligand; 2) the development of high-affinity orthogonal HTP/HTL pairs with minimal cross-reactivity; 3) a rational design of a novel peptide/RNA interaction to promote the avidity of binding and create a “single read” display technology GRIP.2. GRIP Display represents a valuable resource for the protein engineering community, and can substantially advance the range of neurobiological questions amenable to DART.

The molybdenum cofactor (Moco) is an essential enzyme cofactor found in all kingdoms of life. Moco plays central roles in many vital biological processes, and must be biosynthesized de novo. During its biosynthesis, the characteristic pyranopterin ring of Moco is constructed by a complex rearrangement of guanosine 5'-­triphosphate (GTP) into cyclic pyranopterin (cPMP) through the action of two enzymes, MoaA and MoaC. However, the mechanisms and the functions of the two enzymes are under significant debate. To elucidate their physiological roles, I took a multidisciplinary approach to functionally characterize MoaA and MoaC in vivo and in vitro. In this dissertation, I report the first isolation and characterization of the physiological MoaC substrate, 3',8-­ cyclo-­7,8-­dihydro-­guanosine 5'-triphosphate (3',8-cH2GTP). I also report the first X-­ray crystal structures of MoaC in complex with this highly air sensitive substrate, and its product cPMP. These studies, combined with in vitro experiments using substrate analogs, catalytically impaired mutants, and synthetic peptides, have enabled me to delineate the functions of the Moco biosynthetic enzymes, MoaA and MoaC, and proposed mechanistic models for their roles in the formation of cPMP.

In this dissertation we set out to describe the excited state properties of organic molecules as well as the inherent reactivity of organic molecules from a computational perspective. In order to compute excitation energies, we use the particle-particle random phase approximation (pp-RPA) to the pairing matrix fluctuation. We apply the pp-RPA to a set of organic molecules that exhibit thermally activated delayed fluorescence. The charge-transfer excited states are accurately reproduced with the pp-RPA. This class of molecules represent the largest molecules studied with the pp-RPA.

We also present method development to mitigate a shortcoming of the pp-RPA. Previously, the pp-RPA approach to computing excitation energies was limited to describing excitations that originate from the highest occupied molecular orbital (HOMO). We adopt a non-optimized pp-RPA reference, which allowed us to compute the valence excitation energies that originate from any orbital below the HOMO. This approach was applied to a set of benchmark organic molecules.

With respect to molecular transformations, we provide computational insight to the regioselective hydroamination of unsaturated organic molecules with the aid of density functional theory (DFT). Using concepts from conceptual DFT we uncover that the observed regioselectivity is driven by the inherent reactivity of the organic molecule of interest. Our analysis provides insight into the controllable addition of N―H bond across an unsaturated olefin.