The de novo design of peptides is a central area of research in chemical biology. Although it is now possible to design helical peptide structures from first principle, designing â-sheets remains a challenge. Significant advances in this area have been made by using molecular scaffolds, which stabilize â-sheets through intramolecular H-bonding involving the scaffold or which direct supramolecular assembly of the conjugate. In my thesis, I have made use of novel strategies, using ferrocene (Fc) as a central scaffold for controlling the secondary structure of peptides. This approach has been highly successful. Four major new strategies are introduced and described in this thesis: <p>a) Cyclization of Fc-peptide conjugates of the type Fc[CO-Xxx-CSA]2 (Xxx = Gly, Ala, Val, Leu) and Fc[CO-Gly-Xxx-CSA]2 (Xxx = Val, Ile; CSA = cysteamine) leads to the clean formation of novel cyclic bioorganometallic conjugates, which exhibit strong intramolecular hydrogen bonding interactions that restrict the mobility of the podand peptide chains. In the latter system, this intermolecular hydrogen bonding interaction was exploited for the design of a novel â-barrel-like structure. For Fc[CO-Gly-Val-CSA]2 and Fc[CO-Gly-Ile-CSA]2 discrete cyclic supramolecular assemblies were formed in which the individual molecules assemble along the rims of the molecules, resulting in the formation of tubular peptide superstructures that possess a central cavity and are filled with water molecules. <p>b) Prior to my work, work by Hirao and Metzler-Nolte clearly showed that the two podand peptide chains in Fc-peptide conjugates are pointing away from each other. This would indicate that extended â-sheets cannot be formed by simply extending the podand peptide chains. In my work, I clearly demonstrate that, in contrast to earlier results, it is possible to use the Fc scaffold to stabilize â-sheet-like interactions in longer peptide chains. Two systems are described in this thesis Fc[CO-Gly-Val-Cys(Bz)-OMe]2 and Fc[CO-Gly-Ile-Cys(Bz)-OMe]2. In both the cases, amino acids are employed that have a high propensity for â-sheet formation. Both Fc-peptide conjugates exhibit strong interstrand hydrogen bonding, resembling that found in â-sheets.<p>c) In this work, I have demonstrated the use of ferrocene amino acid (Fca) to control the structure in peptides. In contrast to previous work by Metzler-Nolte, my work is largely focusing on the design of a repetitive Fca-peptide motif. It is proposed that this repetition will enable strong interactions between the peptide portions of the conjugate, resulting in the formation of an extended structure. To this effect, a series of Fca-conjugates of the type Boc-[Fca-Ala]n-OMe (n = 1-4) was synthesized and fully characterized. All systems display the expected interaction between the Ala residues having a 12-membered hydrogen bonded ring. Such a structural motif resembles that found in naturally occurring â-helical structures of the spike-region of some viral proteins. <p>d) I have also demonstrated the use of a novel Fc-derivative, Fc[NH-Boc]2, to control the structure of podand amino acid chains. Fc-diamine was synthesized by the convenient carbazide route giving this useful scaffold in high yield. This material was converted into its peptide conjugate and the resulting conjugate displays the elusive 14-membered hydrogen bonding ring. <p>Thus, in my work, I have provided a new complementary tool for peptide design that will undoubtedly find applications for the design of de novo proteins in the near future.

The de novo design of peptides is a central area of research in chemical biology. Although it is now possible to design helical peptide structures from first principle, designing â-sheets remains a challenge. Significant advances in this area have been made by using molecular scaffolds, which stabilize â-sheets through intramolecular H-bonding involving the scaffold or which direct supramolecular assembly of the conjugate. In my thesis, I have made use of novel strategies, using ferrocene (Fc) as a central scaffold for controlling the secondary structure of peptides. This approach has been highly successful. Four major new strategies are introduced and described in this thesis: <p>a) Cyclization of Fc-peptide conjugates of the type Fc[CO-Xxx-CSA]2 (Xxx = Gly, Ala, Val, Leu) and Fc[CO-Gly-Xxx-CSA]2 (Xxx = Val, Ile; CSA = cysteamine) leads to the clean formation of novel cyclic bioorganometallic conjugates, which exhibit strong intramolecular hydrogen bonding interactions that restrict the mobility of the podand peptide chains. In the latter system, this intermolecular hydrogen bonding interaction was exploited for the design of a novel â-barrel-like structure. For Fc[CO-Gly-Val-CSA]2 and Fc[CO-Gly-Ile-CSA]2 discrete cyclic supramolecular assemblies were formed in which the individual molecules assemble along the rims of the molecules, resulting in the formation of tubular peptide superstructures that possess a central cavity and are filled with water molecules. <p>b) Prior to my work, work by Hirao and Metzler-Nolte clearly showed that the two podand peptide chains in Fc-peptide conjugates are pointing away from each other. This would indicate that extended â-sheets cannot be formed by simply extending the podand peptide chains. In my work, I clearly demonstrate that, in contrast to earlier results, it is possible to use the Fc scaffold to stabilize â-sheet-like interactions in longer peptide chains. Two systems are described in this thesis Fc[CO-Gly-Val-Cys(Bz)-OMe]2 and Fc[CO-Gly-Ile-Cys(Bz)-OMe]2. In both the cases, amino acids are employed that have a high propensity for â-sheet formation. Both Fc-peptide conjugates exhibit strong interstrand hydrogen bonding, resembling that found in â-sheets.<p>c) In this work, I have demonstrated the use of ferrocene amino acid (Fca) to control the structure in peptides. In contrast to previous work by Metzler-Nolte, my work is largely focusing on the design of a repetitive Fca-peptide motif. It is proposed that this repetition will enable strong interactions between the peptide portions of the conjugate, resulting in the formation of an extended structure. To this effect, a series of Fca-conjugates of the type Boc-[Fca-Ala]n-OMe (n = 1-4) was synthesized and fully characterized. All systems display the expected interaction between the Ala residues having a 12-membered hydrogen bonded ring. Such a structural motif resembles that found in naturally occurring â-helical structures of the spike-region of some viral proteins. <p>d) I have also demonstrated the use of a novel Fc-derivative, Fc[NH-Boc]2, to control the structure of podand amino acid chains. Fc-diamine was synthesized by the convenient carbazide route giving this useful scaffold in high yield. This material was converted into its peptide conjugate and the resulting conjugate displays the elusive 14-membered hydrogen bonding ring. <p>Thus, in my work, I have provided a new complementary tool for peptide design that will undoubtedly find applications for the design of de novo proteins in the near future.

Proteins have a wide array of essential functions: from serving as enzymatic catalysts to protecting the immune system as antibodies. Proteins spontaneously self-organize into specific, folded structures determined by their amino acid sequences and the interaction between molecular forces. Since the 3-dimensional structure into which they fold often relates to the specific function of the protein, much effort has been directed towards methods to predict the folded structure from a given sequence, with the hope of being able to understand protein functions from sequence information. The protein folding problem can be summarized as the attempt to understand the relationship between a protein sequence and a protein's geometric shape, or fold. Thus, there are two principal problems: given a sequence, what 3-dimensional form will the protein take (forward problem), and given a particular fold, what sequence or sequences code for that form (the inverse problem). In this work, models that represent folds as continuous structures are explored. Models of the two prevalent motifs in protein folds, α helices and β barrels, are developed using axially deformed tubes and surfaces of revolution. These models are then analyzed and used to develop coordinate models of known and unknown structures.

Single-molecule force spectroscopy (SMFS) is a unique approach to study the mechanical unfolding of proteins. SMFS unfolding experiments yield insight into how interactions stabilize a protein and guide its unfolding and refolding pathways. In contrast to various water-soluble proteins whose unfolding and refolding patterns have been characterized, only α-helical membrane proteins have been probed by SMFS. It was shown that α-helical membrane proteins unfold via many intermediates; this differs from the two-state unfolding process usually observed in water-soluble proteins. In membrane proteins, upon mechanically pulling the peptide end of the protein, single and grouped α-helices and polypeptide loops unfold in steps until the entire protein is unfolded. Whether the α-helices and loops unfold individually or cooperatively to form an unfolding intermediate depends on the interactions established within the membrane protein and the membrane. Each unfolding event relates to an unfolding intermediate with the sequence of these intermediates defining the unfolding pathway of the protein. β-barrel-forming membrane proteins are the second major group of membrane proteins and have not yet been studied by SMFS. To fill this void this study was designed to characterize interactions, unfolding, and refolding of the β-barrel forming outermembrane protein G (OmpG).Folding of transmembrane proteins, despite the important part these proteins play in every biological process in a cell, is studied in only a few examples. Of those only a handful were β-stranded membrane proteins (Tamm et al., 2004; Kleinschmidt et al., 2006). Current models describe that transmembrane β-barrels fold into the lipid membrane via two major steps. First the unfolded polypeptide interacts with the lipid surface where it then folds and inserts into the membrane (Kleinschmidt et al., 2006; Huysmans et al., 2010). Conventionally, thermal or chemical denaturation is used to study folding of membrane proteins. In most cases membrane proteins were solubilized in detergent or exposed to urea to be studied, conditions that are not compatible with In vivo conditions. This suggests that the folding pathways described so far may not be a realistic representation of such pathways in nature. SMFS represents a unique approach to study the unfolding and refolding of membrane proteins into the lipid membrane (Kedrov et al., 2006; Kessler et al., 2006). Using SMFS makes it possible to study unfolding and refolding of membrane proteins in their nativephysiological environment with controlled pH, electrolyte, temperature, and most importantly in the absence of any chemical denaturant or detergent. In this thesis, SMFS was utilized to unfold and refold OmpG in E coli lipid extract. Bulk unfolding experiments suggested that OmpG unfolds and folds reversibly and much faster than α-helical proteins (Conlan et al., 2000). The folding process is thought to be a coupled two-state membrane partition-folding reaction. To the contrary, the mechanical unfolding of OmpG consisted of many sequential unfolding intermediates. Our SMFS refolding experiments showed that a partially unfolded OmpG molecule also refolds via several sequential steps. The predominant refolding steps are defined by individual β-hairpins that could later assemble the transmembrane β-barrel of OmpG. In conclusion, the most probable unfolding and refolding pathways of OmpG as a membrane β-barrel protein go through the β-hairpins as the structural segments or unfolding-refolding intermediates and the process is a multi step one rather than the simple two state process. We also used SMFS to study the physical interactions that switch the functional state and gating of OmpG. The structural changes that gate OmpG have been previously described by X-ray crystallography (Yildiz et al., 2006). They showed when the pH changes from neutral to acidic the flexible extracellular loop L6 folds into the pore and closes the OmpG pore. Here, SMFS was used to structurally localize and quantify the interactions that are associated with the pH-dependent closure. At an acidic pH, a pH-dependent interaction at loop L6 was detected. This interaction changed the unfolding of loop L6 and β-strands 11 and 12, which connect loop L6. All other interactions detected within OmpG were found to be unaffected by changes in pH. These results provide a quantitative and mechanistic explanation of how pHdependent interactions change the folding of a peptide loop to gate the transmembrane pore. It has also been shown how the stability of OmpG is optimized so that pH changes modify only those interactions necessary to gate the transmembrane pore and there are no global changes in protein conformation or mechanical properties. In the next step of interactions study, dynamic SMFS (DFS) was applied to quantify the parameters characterizing the energy barriers in energy landscape for unfolding of the OmpG. Some of these parameters are: free energy of activation and distance of the transition state from the folded state. The pH-dependent functional switching of OmpG directs the protein along different regions at the unfolding energy landscape. The two functional states of OmpG sequential folding take the same unfolding pathway as β-hairpins I–IV. After the initial unfolding events, the unfolding pathways diverge. In the open state, the unfolding of β-hairpin V in one step precedes the unfolding of β-hairpin VI. In the closed state, β-hairpin V and β-strand S11 with a part of extracellular loop L6 unfold cooperatively, and subsequently β-strand S12 unfolds with the remaining loop L6. These two unfolding pathways in the open and closed states join again in the last unfolding step of β-hairpin VII. Also, the conformational change from the open to the closed state witnesses a difference in Xu and κ in the energy landscape that translates to rigidified extracellular loop L6 at the gating area. Thus, a change in the conformational state of OmpG not only bifurcates its unfolding pathways but also tunes its mechanical properties for optimum function.

Single-molecule force spectroscopy (SMFS) is a unique approach to study the mechanical unfolding of proteins. SMFS unfolding experiments yield insight into how interactions stabilize a protein and guide its unfolding and refolding pathways. In contrast to various water-soluble proteins whose unfolding and refolding patterns have been characterized, only α-helical membrane proteins have been probed by SMFS. It was shown that α-helical membrane proteins unfold via many intermediates; this differs from the two-state unfolding process usually observed in water-soluble proteins. In membrane proteins, upon mechanically pulling the peptide end of the protein, single and grouped α-helices and polypeptide loops unfold in steps until the entire protein is unfolded. Whether the α-helices and loops unfold individually or cooperatively to form an unfolding intermediate depends on the interactions established within the membrane protein and the membrane. Each unfolding event relates to an unfolding intermediate with the sequence of these intermediates defining the unfolding pathway of the protein. β-barrel-forming membrane proteins are the second major group of membrane proteins and have not yet been studied by SMFS. To fill this void this study was designed to characterize interactions, unfolding, and refolding of the β-barrel forming outermembrane protein G (OmpG).Folding of transmembrane proteins, despite the important part these proteins play in every biological process in a cell, is studied in only a few examples. Of those only a handful were β-stranded membrane proteins (Tamm et al., 2004; Kleinschmidt et al., 2006). Current models describe that transmembrane β-barrels fold into the lipid membrane via two major steps. First the unfolded polypeptide interacts with the lipid surface where it then folds and inserts into the membrane (Kleinschmidt et al., 2006; Huysmans et al., 2010). Conventionally, thermal or chemical denaturation is used to study folding of membrane proteins. In most cases membrane proteins were solubilized in detergent or exposed to urea to be studied, conditions that are not compatible with In vivo conditions. This suggests that the folding pathways described so far may not be a realistic representation of such pathways in nature. SMFS represents a unique approach to study the unfolding and refolding of membrane proteins into the lipid membrane (Kedrov et al., 2006; Kessler et al., 2006). Using SMFS makes it possible to study unfolding and refolding of membrane proteins in their nativephysiological environment with controlled pH, electrolyte, temperature, and most importantly in the absence of any chemical denaturant or detergent. In this thesis, SMFS was utilized to unfold and refold OmpG in E coli lipid extract. Bulk unfolding experiments suggested that OmpG unfolds and folds reversibly and much faster than α-helical proteins (Conlan et al., 2000). The folding process is thought to be a coupled two-state membrane partition-folding reaction. To the contrary, the mechanical unfolding of OmpG consisted of many sequential unfolding intermediates. Our SMFS refolding experiments showed that a partially unfolded OmpG molecule also refolds via several sequential steps. The predominant refolding steps are defined by individual β-hairpins that could later assemble the transmembrane β-barrel of OmpG. In conclusion, the most probable unfolding and refolding pathways of OmpG as a membrane β-barrel protein go through the β-hairpins as the structural segments or unfolding-refolding intermediates and the process is a multi step one rather than the simple two state process. We also used SMFS to study the physical interactions that switch the functional state and gating of OmpG. The structural changes that gate OmpG have been previously described by X-ray crystallography (Yildiz et al., 2006). They showed when the pH changes from neutral to acidic the flexible extracellular loop L6 folds into the pore and closes the OmpG pore. Here, SMFS was used to structurally localize and quantify the interactions that are associated with the pH-dependent closure. At an acidic pH, a pH-dependent interaction at loop L6 was detected. This interaction changed the unfolding of loop L6 and β-strands 11 and 12, which connect loop L6. All other interactions detected within OmpG were found to be unaffected by changes in pH. These results provide a quantitative and mechanistic explanation of how pHdependent interactions change the folding of a peptide loop to gate the transmembrane pore. It has also been shown how the stability of OmpG is optimized so that pH changes modify only those interactions necessary to gate the transmembrane pore and there are no global changes in protein conformation or mechanical properties. In the next step of interactions study, dynamic SMFS (DFS) was applied to quantify the parameters characterizing the energy barriers in energy landscape for unfolding of the OmpG. Some of these parameters are: free energy of activation and distance of the transition state from the folded state. The pH-dependent functional switching of OmpG directs the protein along different regions at the unfolding energy landscape. The two functional states of OmpG sequential folding take the same unfolding pathway as β-hairpins I–IV. After the initial unfolding events, the unfolding pathways diverge. In the open state, the unfolding of β-hairpin V in one step precedes the unfolding of β-hairpin VI. In the closed state, β-hairpin V and β-strand S11 with a part of extracellular loop L6 unfold cooperatively, and subsequently β-strand S12 unfolds with the remaining loop L6. These two unfolding pathways in the open and closed states join again in the last unfolding step of β-hairpin VII. Also, the conformational change from the open to the closed state witnesses a difference in Xu and κ in the energy landscape that translates to rigidified extracellular loop L6 at the gating area. Thus, a change in the conformational state of OmpG not only bifurcates its unfolding pathways but also tunes its mechanical properties for optimum function.:Table of Contents INTRODUCTION:1 1.1 THE FIRST UNIT OF LIFE STARTED WITH MEMBRANE:1 1.2.1 CELL MEMBRANE STRUCTURE: 2 1.3 MEMBRANE PROTEINS:3 1.3.1 α-­‐HELICAL MEMBRANE PROTEINS:5 1.3.2 β-­‐BARREL MEMBRANE PROTEIN:5 1.4 MEMBRANE PROTEINS FOLDING:12 1.4.1 MODELS FOR α-­‐HELICAL MEMBRANE PROTEIN FOLDING:13 1.4.2 MODELS FOR β-­‐BARREL MEMBRANE PROTEIN FOLDING:15 1.5. GATING STUDY OF MEMBRANE PROTEINS:18 ATOMIC FORCE MICROSCOPY:19 2.1 ATOMIC FORCE MICROSCOPE:19 2.1.1 HISTORY:19 2.1.2 PRINCIPLE:19 2.1.3 THE CANTILEVER:20 2.1.4 AFM MODES 23 2.2 SINGLE-­‐MOLECULE FORCE SPECTROSCOPY:25 2.2.1 DYNAMIC FORCE SPECTROSCOPY,(DYNAMIC SMFS):27 2.3 WHAT IS THE ADVANTAGE OF USING ATOMIC FORCE MICROSCOPY IN MEMBRANE PROTEIN STUDIES?:29 FOLDING MECHANISM OF OMPG:31 3.1 UNFOLDING PATTERN: ONEβ-­‐HAIRPIN AFTER THE OTHER:31 3.1.1 OUTER MEMBRANE PROTEIN G (OMPG):31 3.1.2 MECHANICAL UNFOLDING PATHWAYS OF THE MEMBRANE β-­‐BARREL PROTEIN OMPG:33 3.1.3 MATERIAL AND METHODS:34 3.1.4 RESULTS AND DISCUSSION:41 3.2 REFOLDING PATTERN: ONE Β-­‐HAIRPIN AFTER THE OTHER:48 3.2.1. EXPLORING REFOLDING PATHWAYS AND KINETICS OF THE MEMBRANE Β-­‐BARREL PROTEIN OMPG:48 3.2.2 EXPERIMENTAL PROCEDURES:49 3.2.3 RESULTS:50 3.2.4 DISCUSSION:52 INTERACTION STUDIES:59 4.1 PH-­‐DEPENDENT INTERACTIONS GUIDE THE FOLDING AND GATE THE TRANSMEMBRANE PORE OF THE β-­‐BARREL TRANSMEMBRANE PROTEIN OMPG:59 4.1.2 INTRODUCTION:59 4.1.2 EXPERIMENTAL PROCEDURES:61 4.1.3 RESULTS AND DISCUSSION:62 4.2 DUAL ENERGY LANDSCAPE: THE FUNCTIONAL STATE OF THE OUTER MEMBRANE β-­‐BARREL PROTEIN OMPG MOLDS ITS UNFOLDING ENERGY LANDSCAPE:67 4.2.1 INTRODUCTION:67 4.2.2 EXPERIMENTAL PROCEDURES:71 4.2.3 RESULTS AND DISCUSSION:74 4.2.3.1 FUNCTIONAL STATE OF OMPG DIRECTS ITS UNFOLDING ROUTE:74 4.2.3.2 QUANTIFYING THE UNFOLDING ENERGY BARRIERS OF OMPG IN THE CLOSED AND OPEN CONFORMATIONS:75 4.2.3.3 TRANSITION STATE DISTANCES OF UNFOLDING ENERGY BARRIERS:77 4.2.3.4 ACTIVATION FREE ENERGY OF Β-­‐STRANDS AND Β-­‐HAIRPINS:79 4.2.3.5 MECHANICAL PROPERTIES OF OMPG:83 4.2.3.6 MAPPING THE UNFOLDING ENERGY LANDSCAPES OF OMPG IN THE OPEN AND CLOSED STATES:85 4.2.4 CONCLUSION:86 OUTLOOK:89 5.1 INTRODUCTION:89 5.2 INTERACTION STUDY AND UNFOLDING ENERGY LANDSCAPE:90 5.3 MEMBRANE PROTEINF OLDING:92 REFRENCES:96 ABBREVIATIONS:110 SYMBOLS:111 PUBLICATIONS:113 ACKNOWLEDGMENT:114 DECLARATION: 115

Rare lipoprotein A (RlpA) has been studied previously only in Escherichia coli, where it localizes to the septal ring and scattered foci along the lateral wall, but mutants have no phenotypic change. In this thesis, we show rlpA mutants of Pseudomonas aeruginosa form chains of short, fat cells when grown in media of low osmotic strength. These morphological defects indicate RlpA is needed for efficient separation of daughter cells and maintenance of rod shape. Analysis of peptidoglycan sacculi from a ΔrlpA mutant revealed increased tetra and hexasaccharides that lack stem peptides (hereafter called "naked glycans"). Incubation of these sacculi with purified RlpA resulted in release of naked glycans containing 1,6-anhydro N-acetylmuramic acid ends. RlpA did not degrade sacculi from wild-type cells unless the sacculi were subjected to a limited digestion with an amidase to remove some of the stem peptides. Collectively, these findings indicate RlpA is a lytic transglycosylase with a strong preference for naked glycan strands. We propose that RlpA activity is regulated in vivo by substrate availability, and that amidases and RlpA work in tandem to degrade peptidoglycan in the division septum and lateral wall. Our discovery that RlpA from P. aeruginosa is a lytic transglycosylase motivated us to reinvestigate RlpA from E. coli. We confirmed predictions that RlpA of E. coli is an outer membrane protein and determined its abundance to be about 600 molecules per cell. However, multiple efforts to demonstrate that E. coli RlpA is a lytic transglycosylase were unsuccessful and the function of this protein in E. coli remains obscure.

β-barrel membrane proteins play important structural and functional roles in Gram negative bacteria and in mitochondria and chloroplasts of eukaryotes. A conserved machine is responsible for the folding and insertion of β-barrel membrane proteins but its mechanism remains largely unknown. In E. coli, a five protein β-barrel assembly machine (Bam) assembles β-barrel proteins into the outer membrane (OM). Among all β-barrel membrane proteins in E. coli , the β-barrel component of the OM LPS translocon is one of only two essential β-barrels, the other being the central component of the Bam machinery itself. The OM LPS translocon, which consists of OM β-barrel protein LptD (lipopolysaccharide transport) and OM lipoprotein LptE, is responsible for the final export of LPS molecules into the outer leaflet of the OM, resulting in an asymmetric bilayer that blocks the entry of toxic molecules such as antibiotics. This thesis describes the characterization of the biogenesis pathway of the OM LPS translocon and its folding and insertion into the OM by the Bam machinery. An in vivo S35-Methionine pulse-labeling assay was developed to identify intermediates along the biogenesis of the OM LPS translocon. Seven intermediates were identified along the pathway. We show that proper assembly of the OM LPS translocon involves an oxidative disulfide bond rearrangement from a nonfunctional intermediate containing non-native disulfides. We also found that the rate determining step of OM LPS translocon biogenesis is β-barrels folding process by the Bam machinery. Using in vivo chemical crosslinking, we accumulated and trapped a mutant form of LptD on BamA, the central component of the Bam machinery. We extended the S35-Methionine pulse-labeling method to allow chemical crosslinking of substrates on the Bam complex and trapped LptD while it was being folded on the Bam machine. We demonstrated that the interaction between LptD and BamA is independent of LptE, while that between LptD and BamD, the other essential component of the Bam complex beside BamA, is LptE dependent. Based on these findings, we proposed a model of Bam-assisted folding of the OM LPS translocon in which LptE templates the folding of LptD. / Chemistry and Chemical Biology

Alzheimer's Disease (AD) is a devastating neurodegenerative disease that is characterized by brain atrophy, neuronal and synaptic loss, cognitive decline, trouble handling activities of daily life, and ultimately leads to death. Worldwide, at least 30 million people suffer from AD, with 5.8 million suffering in the US alone. Despite extensive basic and clinical research, the underlying molecular mechanisms behind AD remain largely unknown. There are four FDA-approved compounds are used for alleviating symptoms but have no curative potency. The first potentially disease-modifying AD drug, aducanumb, was approved by FDA in June 2021. The main histopathological traits of AD are the Amyloid-beta (Aβ) peptide and the tau protein. Aβ aggregates to form extracellular plaques in brain parenchyma and vasculature while tau forms intraneuronal tangles. Aβ is produced by enzymatic cleavage of the amyloid precursor protein (APP) in the brain. Once APP cleavage occurs, Ab monomers either aggregate extracellularly to form buildups of sticky plaque or embed themselves within the neuronal cell membrane to form pores, causing homeostatic dysregulation and eventually cell death. The mechanism of membrane pores formed by Ab and the pore structure remain to be characterized. This study aims to analyze the structure of four Aβ species in lipid membranes. These are the most abundant form of Aβ, Aβ1-40, and the more cytotoxic form, Aβ1-42, as well as their pyroglutamylated counterparts, pEAβ3-40 and pEAβ3-42, which are hypertoxic. These peptides have been studied using biophysical approaches, i.e., circular dichroism, fluorescence spectroscopy, and Fourier transform infrared spectroscopy. Elucidation of the structure of Aβ membrane pores provides valuable insight into the mechanism of Aβ toxicity and may help develop novel therapies for the lethal mystery that is AD.

High-throughput genomics projects have resulted in a rapid accumulation of protein sequences. Therefore, computational methods that can predict protein functions and functional sites efficiently and accurately are in high demand. In addition, prediction methods utilizing only sequence information are of particular interest because for most proteins, 3-dimensional structures are not available. However, there are several key challenges in developing methods for predicting protein function and functional sites. These challenges include the following: the construction of representative datasets to train and evaluate the method, the collection of features related to the protein functions, the selection of the most useful features, and the integration of selected features into suitable computational models. In this proposed study, we tackle these challenges by developing procedures for benchmark dataset construction and protein feature extraction, implementing efficient feature selection strategies, and developing effective machine learning algorithms for protein function and functional site predictions. We investigate these challenges in three bioinformatics tasks: the discovery of transmembrane beta-barrel (TMB) proteins in gram-negative bacterial proteomes, the identification of deleterious non-synonymous single nucleotide polymorphisms (nsSNPs), and the identification of helix-turn-helix (HTH) motifs from protein sequence.

<p dir="ltr">The obligate human pathogen, <i>Neisseria gonorrhoeae </i>(Ngo), has continued to acquire widespread antibiotic resistance. Ngo is the causative agent of the sexually transmitted disease gonorrhea, and can cause additional complications such as endocarditis, septicemia, and infertility if left untreated. The Centers for Disease Control and Prevention (CDC) now recommends a treatment option of a single drug of last resort, ceftriaxone, leaving a need for novel therapeutics against this pathogen.</p><p dir="ltr">Like many bacterial pathogens, Ngo is Gram-negative consisting of both an inner membrane (IM) and outer membrane (OM). The transmembrane proteins in the IM have primarily an α-helical fold, while the transmembrane proteins in the OM have a β-barrel fold. These β-barrel outer membrane proteins (OMPs) have essential functions in regulating the homeostasis and nutrient acquisition of the cell, in addition to promoting virulence in pathogenic strains. These OMPs are folded and inserted into the outer membrane by the β-barrel assembly machinery (BAM) complex. In <i>E. coli,</i> BAM consists of five proteins: BamA, an OMP itself, and four lipoproteins, BamB, C, D, and E.</p><p dir="ltr">Here we present our work toward the structural characterization of BAM from Ngo (<i>Ng</i>BAM) using cryo-EM. Ngo lack a homolog of BamB and may function as a four component complex. To better understand the mechanism for how <i>Ng</i>BAM is able to mediate OMP biogenesis despite lacking a component that is critical in <i>E. coli</i>, we determined the cryo-EM structure of <i>Ng</i>BAM, which revealed several distinct features including that the barrel domain of BamA being observed in the inward-open conformation. We also investigated <i>Ng</i>BAM as a therapeutic target, by studying its interaction with a novel broad spectrum antibiotic darobactin. We first showed darobactin is effective against the laboratory strains of NgoFA19 and ATCC-49226. We also show it is effective against the human isolate WHOX, with a comparable MIC to ceftriaxone. To structurally characterize the mechanism of inhibition by darobactin, we used cryo-EM to determine the structures of <i>Ng</i>BAM bound to two darobactin compounds. In these structures, darobactin binding was accompanied by large conformational changes in <i>Ng</i>BamA. To further probe the effects of darobactin on the conformational plasticity of <i>Ng</i>BAM we performed experiments using double electron-electron resonance spectroscopy, which showed distance changes between the engineered site labels consistent with the conformational changes observed in our structural observation. In addition, narrowing of the peak distributions indicated that darobactin binding was reducing the overall conformational heterogeneity of the complex. Taken together, the work presented here contributes to the understanding of how <i>Ng</i>BAM functions in folding and inserting OMPs and provides a foundation for future structure based drug design of darobactin and other potential compounds.</p>