<p> Human apolipoprotein A-I (apoA-I) has been shown to exhibit antimicrobial activity by neutralizing lipopolysaccharides and destabilizing inner membranes of gram-negative bacteria. Previous studies showed that acrolein, a highly reactive &alpha;&beta; unsaturated aldehyde an environmental pollutant and an endogenously generated lipid peroxidation product, modifies &epsiv;-amino groups of lysine residues in apoA-I. The current study investigated the effect of acrolein exposure on the structure and antimicrobial activity of apoA-I. Acrolein modification was evident by the appearance of apoA-I oligomers due to intermolecular crosslinking, as well as reactivity with an antibody specific for acrolein. Increase of the acrolein to protein ratio resulted in heavily cross-linked apoA-I. Circular dichroism analysis showed that the &alpha;-helical content of acrolein-modified apoA-I was decreased but the protein retained &alpha;-helicity. The stability of modified proteins was increased. Phosphatidylglycerol binding was strongly affected when apoA-I was modified with acrolein. Lipopolysaccharide binding experiments showed that the binding of acrolein modified apoA-I became weaker than unmodified apoA-I. These results suggest that apoA-I modification by acrolein leads to decreased binding of apoA-I to bacterial membranes making it less potent as an antimicrobial protein.</p><p>

<p> Apolipoprotein A-I (apoA-I) is an exchangeable apolipoprotein that constitutes the major protein component of high density cholesterol. ApoA-I is a two-domain protein comprising an N-terminal helix bundle and a less-structured C-terminal domain in the lipid-free state. In the present study, the contribution of the C-terminal domain to the lipid binding and antimicrobial activity of apoA-I was investigated using a chimeric construct in which the C-terminal domain of apoA-I (179-243) was attached to an insect apolipoprotein, <i> Locusta migratoria</i> apolipophorin III (apoLp-III), bearing cysteine substitutions for residues 20 and 149. Circular dichroism results were consistent with the addition of a poorly structured domain to apoLp-III and revealed the apoLp-III helix bundle was successfully closed under oxidizing conditions. Electrophoresis, fluorescence spectroscopy and an in vitro study using macrophage cells revealed that the C-terminal domain in itself was insufficient for efficient binding to lipid, lipopolysaccharide and phosphatidylglycerol vesicles. These results suggest the underlying mechanisms governing these interactions are potentiated by cooperativity between the N- and C-terminal domains of apoA-I. </p>

To maximize the probability of obtaining an antibody peptidase, an efficient assay for the hydrolysis of peptide bonds was developed. Two methods for detecting the uncatalyzed rate of hydrolysis of a peptide bond, without the use of extreme temperature or pH, are described. The uncatalyzed and carboxypeptidase-catalyzed hydrolysis of hippurylphenylalanine was followed under identical conditions by derivatization of the primary-amine product with naphthalenedicarboxaldehyde (NDA). The half-life of this peptide as well as the rate enhancement and catalytic proficiency of carboxypeptidase A were determined. Progress was made in monitoring the hydrolysis of peptidyl-prolyl bonds by derivatization of the secondary-amine product with 4-(dimethylamino)azobenzene-4$\sp\prime$-sulfonyl (dabsyl) chloride. The effect of torsional strain on the rate of peptide-bond hydrolysis was also investigated. Antibodies were elicited against FK520, a hapten that mimics a twisted peptide bond in addition to the transition state for peptide-bond hydrolysis. The six monoclonal antibodies obtained were characterized by competitive ELISA. The binding of FK520 to the antibodies is specifically inhibited by FK506 hut not by rapamycin, indicative that the antibodies do not recognize the $\alpha$-ketoamide functionality of FK520. Nonetheless, the panel of antibodies characterized may prove useful in future studies of immunusuppression.

The essential features of chemotactic signaling in E. coli include: high sensitivity over a wide dynamic range of chemoattractant concentrations, ability to integrate signals from different types of chemoreceptors, and a high gain. It is mediated by a central kinase, CheA, and an adaptor protein, CheW, which form noncovalent complexes with clusters of transmembrane receptors. In this study the molecular bases of these important features were elucidated utilizing functional complexes of template-assembled cytoplasmic fragments (CFs) of a chemoreceptor: Tar, Tsr or Trg (aspartate, serine or ribose/galactose receptors, respectively). Template-assembled complexes made with the wildtype CF (wt-CF) activated CheA, whereas complexes formed with any one of a number of inactivating point mutant CFs (in-CFs) did not. Measurements of kinase stimulation and complex formation revealed different mechanisms of kinase regulation involving competitive and cooperative interactions among receptor fragments. The apparent strength of the cooperative interactions and the composition of half-maximal inhibition (and binding) increased with adaptational covalent modification on the CF. Moreover, cooperative interactions between CFs from receptors of different ligand-binding specificities were also revealed. These interactions occur among CFs of high-abundance (HA) receptors (Tsr and Tar) and low-abundance (LA) receptor (Trg). There was also evidence of functional assistance to LA receptor Trg by HA receptors Tsr and Tar. Receptor fragment clustering was probed by fluorescence resonance energy transfer (FRET). It was shown that receptor clustering is highly correlated with kinase stimulation and complex formation. On the other hand, CheW and CheA were not essential in forming tightly packed receptor clusters, but promoted the process at low densities of the CF-tethering lipid. Covalent modification affected the stability of forming tight clusters and modulated the disrupting effects of some kinase-inactivating CFs to receptor fragment clustering. The clustering states of the wt-CFs from the different receptor types of E. coli also provided an explanation for the conserved natural abundance of these receptors in the cell. Overall, this study has shown the feasibility and utility of the template assembled system in probing functional interactions among proteins involved in bacterial chemotaxis signaling and has contributed to a better understanding of the essential features of this pathway.

Transmembrane signaling events in both eukaryotic and prokaryotic cells occur in the two-dimensional space defined by the lipid membrane. Cellular responses are regulated through processes that involve the recruitment of specific signaling proteins to the membrane surface. These phenomena suggest the importance of clustering and 2D concentration of membrane-bound receptor complexes in signaling. Previously a template-directed method was used to assemble and restore biochemical activity to the cytoplasmic signaling proteins of the E. coli chemotaxis system using a histidine-tagged cytoplasmic fragment of the aspartate receptor (CF) and Ni-NTA lipid vesicles (Shrout et al. 2003). Here, we employed this approach to vary the 2D concentration of CF, which is influenced both CheA activation and methylation. Our data showed that rates of templated CF methylation and the activity of CheA complexes both increased substantially when the 2D concentration of CF was increased. The differing influences of CF density on CheA activation and CF methylation provide a means to regulate CheA activity through receptor clustering.

Tight regulation of caspase-9, a key initiator of apoptosis, is required to uphold cellular homeostasis. Although it is controlled on a multifactorial level, misregulation of this process does occur, which is a characteristic of a variety of diseases from ischemic injury to cancer. Therefore it remains important to gain a detailed understanding of the mechanisms behind native caspase-9 regulatory pathways and harness these mechanisms for therapeutic purposes. Based on known mechanisms, such as the unique inhibitory complex of caspase-9 and XIAP-BIR3, development of synthetic regulators can be envisioned, while other mechanisms such as zinc-mediated inhibition and CARD activation of caspse-9 remain undefined. Intrigued by the multiple ways to control caspase-9's activity, we sought after designing synthetic caspase-9 inhibitors in addition to defining the mechanistic details metal regulation and CARD domain activation. We report the first stabilized &agr;-helical peptides that harness the native regulatory mechanism of caspase-9 and the BIR3 domain which lead to the understanding of the importance of exosites in inhibitory complexes. Our studies also revealed that there are two distinct zinc binding sites, one at the active site and another at a novel zinc binding site of yet unknown function in caspase-9 however this site may have the potential to control caspase-6 based on its regulatory mechanism. Furthermore, an interaction was discovered between CARD and the catalytic core of caspase-9 in the presence of a properly formed substrate binding groove, a potential mechanism utilized by the apoptosome for activation of the enzyme. All in all, the regulation of caspase-9 occurs on a variety of levels that requires almost every surface of the enzyme. Through exploring these underlying molecular details behind the various mechanisms, not only has the field of caspase-9 regulation mechanisms been extended, essential information was gained for further pursuit in an advancement towards the design of caspase-9 activators and inhibitors.

Caspases are cysteine proteases best known for their controlling roles in apoptosis and inflammation. Caspase-6 has recently been shown to play a key role in the cleavage of neurodegenerative substrates that causes Huntington and Alzheimer's Disease, heightening interest in caspase-6 and making it a drug target. All thirteen human caspases have related specificities for binding and cleaving substrate, so achieving caspase-specific regulation at the active site has been extremely challenging if not impossible. We have determined the structures of four unliganded forms of caspase-6, which attain a novel helical structure not observed in any other caspases. In this conformation, rotation of the 90's helix results in formation of a cavity that can function as an allosteric site, locking caspase-6 into an inactive conformation. We are using this cavity to look for chemical ligands that target this cavity and maintain caspase-6 in the inactive, helical conformation. We found that known allosteric inhibitors of caspase-3 and -7 also inhibit caspase-6 through a cavity at the dimer interface. We have determined new structures of a phosphomimetic state and a zinc-bound conformation of caspase-6, which show the molecular details of two additional allosteric sites. The phosphomimetic form of caspase-6 inactivates caspase-6 by disrupting formation of the substrate binding-groove by steric clash of the phosphorylated residue with P201 in the L2' loop. Another allosteric site was found on the "back" of caspase-6 that coordinates a zinc molecule that leads to inactivation. In total we have uncovered four independent allosteric sites in caspase-6, structurally characterized inhibition from these sites and demonstrated that each of these sites might be targeted for caspase-6 specific inhibition by synthetic or natural-product ligands.

Polyketides are a broad class of natural products that have received attention from the scientific community because they are a rich mine of bioactive structures. The common thread that binds the class together is the method by which they are synthesized, by large enzymatic complexes called polyketide synthases which display assembly line like organization. A great deal of effort has been put into studying PKSs, but their mechanistic steps are still not perfectly understood. In order to further the study of PKSs and their components, we have developed a series of reactive small molecules that covalently modify specific sites of PKS components. We have shown that β-lactones are able to selectively load holo-acyl carrier proteins with polyketide-like functionality while leaving similarly functionalized components such as the ketosynthase domain virtually untouched. We have also shown that β-lactams are able discriminate between apo- and holo-ACPs and have used this difference in reactivity to develop a method of purifying holo-ACPs. Another β-lactam based probe has been shown to directly modify the active portion of holo-ACPs with malonate-like functionality, and may be starting point for introducing novel functionality into difficult to access sites of polyketides. During the course of these investigations the copper catalyzed azide-alkyne cycloaddition has been used heavily for conjugation reactions involving small molecules and proteins. The presence of copper has been a serious problem, often leading to lost and damaged proteins. Copper-free azide-alkyne conjugation methods exist, but suffer from drawbacks of their own such as slow reaction rates and difficult targetability. In order to maintain the positive aspects of copper free click yet overcome the drawbacks, we have been developing photosensitive probes which upon irradiation rapidly react with azides in a click like reaction. Although this reaction was initially developed with bioconjugation in mind, further experiments have indicated that this may not be the best use. However, experiments are currently being carried out to optimize their performance and explore their usefulness in other chemical conjugation applications.

Covalent labeling and mass spectrometry are seeing increased use together as a way to obtain insight into the 3-dimensional structure of proteins and protein complexes. Several amino acid specific (e.g., diethylpyrocarbonate) and non-specific (e.g., hydroxyl radicals) labeling reagents are available for this purpose. Diethylpyrocarbonate (DEPC) is a promising labeling reagent because it can potentially probe up to 30% of the residues in the average protein and gives only one reaction product, thereby facilitating mass spectrometric analysis. It was recently reported, though, that DEPC modifications are labile for some amino acids. This dissertation focuses on the improvement of diethylpyrocarbonate (DEPC)-based covalent labeling for increased protein structural resolution. The number of DEPC modified residues and, thus, protein structural information, can be significantly increased by decreasing the time between the covalent labeling reaction and the mass spectrometric analysis. This is most effectively accomplished using short (e.g., 2 h) proteolytic digestions with enzymes such as immobilized chymotrypsin or Glu-C rather than using methods (e.g., microwave or ultrasonic irradiation) that accelerate proteolysis in other ways. Besides, Cys residues that form disulfide bonds appear to be modified by DEPC as well. We demonstrate that disulfide linked Cys residues are not actually reactive with DEPC but, instead, once reduced, free Cys residues can capture a carbethoxy group from other modified amino acids via a solution-phase reaction that can occur during the protein digestion step. This "scrambling" of carbethoxy groups decreases the amount of modification observed at other residues and can potentially provide incorrect protein structural information. Fortunately, label scrambling can be completely avoided by alkylating the free thiols after disulfide reduction. We also developed novel methods as indicators of protein structural integrity using isotopically encoded labeling reagent Tandem Mass Tags. Because most covalent labels are relatively large, steps must be taken to ensure the structural integrity of the modified protein during the labeling reactions so that correct structural information can be obtained. Measuring labeling kinetics is a reliable way to ensure that a given labeling reagent does not perturb a protein's structure, but obtaining such kinetic information is time and sample intensive because it requires multiple liquid chromatography (LC)–MS experiments. Here we present a new strategy that uses isotopically encoded labeling reagents to measure labeling kinetics in a single LC–MS experiment. We illustrate this new strategy by labeling solvent-exposed lysine residues with commercially available tandem mass tags. After tandem MS experiments, these tags allow the simultaneous identification of modified sites and determination of the reaction rates at each site in a way that is just as reliable as experiments that involve multiple LC-MS measurements. This improved technique is then applied to study the structural change of proteins that undergo forced degradation. Because covalent labeling combined with MS can be used to monitor conformational changes of proteins, it can also be applied to study structural changes of proteins after exposure to degradation conditions. β2m was used as a model protein to examine the potential of this method. Forced degradation studies using thermal and oxidative conditions were carried out. Covalent modification patterns of the protein show clear differences under these degradation conditions as compared to the native protein. The general trend that we found was that less modification was observed for both thermal and oxidative condition, indicating that aggregation could occur at higher temperatures or in the presence of hydrogen peroxide. Size exclusion chromatography confirmed that there was dimer formation during the forced degradation conditions, which is consistent with the covalent labeling data. This method could be applied to other systems to study degradation caused structural changes of proteins as it is a fast and relatively simple approach.

Clostridial botulinum neurotoxins (BoNTs) cause neuroparalysis by blocking neurotransmitter release. The toxin's light chain (LC) acts as a Zn2+-endopeptidase. Type A botulinum nuerotoxin light chain (BoNT/A LC) was expressed in E. coli to high yield level. The recombinant BoNT/A LC, which retained the proteolytic activity and secondary structure, was used in this study. During the translocation of the LC to the cytosol, it is exposed to the endosomal low pH. Low pH showed a dramatic change in the BoNT/A LC polypeptide folding as indicated by differential heat denaturation. Furthermore, binding of 1-anilinonaphthalenesulfonate revealed exposure of hydrophobic domains of BoNT/A LC at low pH. Low pH induced changes were completely reversible. Fluorescence measurements suggested that the two Trp residues are buried and constrained in a hydrophobic environment. The reversibility rules out a possible prerequisite of acid treatment of LC for an ultimate functional conformation. BoNT/A is a zinc-endopeptidase that contains the consensus sequence HEXXH in LC. Substitution of Glu-224 in the motif with Gln (E224Q) resulted in a total loss of the endopeptidase activity, whereas substitution with Asp (E224D) retained ∼1% of the enzymatic activity. However, k m values for wild type and E224D LC were similar. Global structure, Zn2+ content, and substrate binding ability are retained in the mutants. Titration of Zn2+ to EDTA-treated wild-type and mutant proteins indicated similar Zn2+ binding affinity constants. These results suggest an essential and direct role of the carboxyl group of Glu-224 in the hydrolysis of the substrate. The location of the carboxyl group at a precise position is critical for the enzymatic activity. BoNT contains one Zn2+/molecule. The active-site Zn 2+, which was easily displaced from the active site by ethylenediaminetetraacetate, reversibly binds to the LC in a stoichiometric manner. Enzymatic activity was completely abolished in zinc-depleted LC (apo-LC). However, Zn 2+ replenishment partially restored the activity in reZn2+ -LC. Comparable Km values in holo- and reZn2+-LC were observed, indicating a similar substrate binding ability. Removal of the zinc causes irreversible tertiary structural change while the secondary structure remains unchanged. Zinc-binding leads to enhanced thermal stability of LC, which is not identical in native holo-LC and reZn 2+-LC.