Browsing by Subject "Peptides"

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Aromatic donor-acceptor interactions : bridging abiotic and peptide folding
(2008-05) Bradford, Valerie Jean, 1980-; Iverson, Brent L.
Aromatic donor-acceptor interactions have been utilized by the Iverson group in the development of abiotic molecules, called aedamers, that achieve new folding motifs, intermolecular association in heteroduplexes, and new material properties. These molecules exploit the interaction between the electron-rich 1,5-dialkoxynapthalene (DAN) and electron-deficient 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) units in a face-centered stacking geometry in aqueous solution. This dissertation describes the use of DAN-NDI interactions in the realm of peptides and proteins to expand the scope for applications of this interaction. This work specifically focuses on three areas of aromatic donor-acceptor interactions: achieving protein behavior with abiotic molecules, introducing the interaction into natural peptides, and utilizing the interaction in the intermolecular association of an abiotic molecule and a natural peptide. Chapter 2 refines the model of aggregation of an amphiphilic aedamer, which forms a hydrogel upon heating. The aedamer behaves similarly to proteins called amlyoids, which form fibrils and plaques in vivo which have been implicated in a variety of diseases, including Alzheimer's. Chapter 3 describes the synthesis of [alpha]-amino acids with DAN- and NDI-containing side chains. These amino acids can be used in a peptide model of [beta]-hairpin secondary structure. The model system can determine whether aromatic donor-acceptor interactions are useful in stabilizing peptide and protein structure. Chapter 4 describes the study of the Anchored Periplasmic Expression System (APEx) for use in screening random peptide libraries. A random peptide library is used to determine the sequence of a natural peptide, potentially containing electron-rich aromatic residues, which could bind an NDI oligomer with high affinity for use as a protein expression tag. Chapter 5 describes work toward the use of cyclic NDI bisintercalators for binding both the major and minor grooves of a specific sequence of DNA simultaneously, in addition to the use of cyclic NDI and DAN molecules for the further study of NDI-DAN interactions in abiotic intermolecular assembiles. Overall, this work has advanced the application of aromatic donor-acceptor interactions in peptides and should serve as a foundation for the future study of this interaction in protein folding and behavior in biological systems.
Biomolecule interactions on calcium carbonate and stoichiometrically similar biomedical, optical and electronic materials
(2004) Gooch, Erin Elaine; Belcher, Angela M.
A combinatorial approach has been successfully used to characterize peptides that bind to four different surfaces of CaCO3 and six different oxide substrates that are chemically and stoichiometrically similar to CaCO3. A standard screening and a single substrate screening method were employed. Both methods used a bacteriophage combinatorial library with a complexity of 109 different random sequences. While geological (104) calcite screenings do not seem to show strong consensus, peptides screened against geological aragonite exhibit extraordinary consensus. Overall, peptides screened against aragonite were highly basic. Peptides screened against (110) geological aragonite show [LAIVG]P[WF][RKH] and triple [RKH] patterns. The most significant binding peptide, A21 (LPPWKHKTSGVA) was found in 4 separate screenings. The (110) geological aragonite sequences are highly enriched in prolines. Coincidently, peptides screened against the optical material LiNbO3 +Z show patterns similar to those found in the aragonite screenings. Other substrates that did not exhibit strong consensus include powdered LiNbO3, powdered BaTiO3, powdered PbTiO3, single crystal LiNbO3 –Z, and hydroxyapatite. A database and analysis program was written to catalog and evaluate the statistical significance of the many sequences. One peptide that was determined to be extremely significant in binding to aragonite, A20 (LPKWQERQMLSA), was modeled using umbrella sampling molecular dynamics techniques as well as analyzed by NMR. It was determined that there is a good probability that the peptide’s conformation is helical, but it is able to introconvert between α-helical, 3-10 helical and extended conformations fast enough that a stable secondary structure is not detectable by NMR. Engineered phage were constructed to display these significant peptides on the pVIII surface, increasing the expression level and the long range order in the hopes of building an aragonite nucleation surface. Hybrid organic-inorganic materials were grown on the phage resulting in a mixture of the three forms of CaCO3: calcite, vaterite, and aragonite. In addition, hybrid materials were grown on the optical waveguide material LiNbO3. Doubly engineered phage were shown to bind preferentially to the LiNbO3 +Z surface through the interaction of the A1 displayed peptide as well as nucleate semiconducting ZnS particles via the A7 peptide selected previously against ZnS.
Characterization and applications of the twin-arginine transporter pathway
(2007-12) Strauch, Eva-Maria, 1979-; Georgiou, George
The twin-arginine translocase allows the translocation of folded protein substrates across the cytoplasmic membrane of bacteria and archaea or the thylakoid membrane of plants. In Escherichia coli, its protein components TatA, TatB and TatC assemble dynamically upon interaction with protein substrates. Prior to export, the machinery performs a quality control check so that only correctly folded proteins are translocated. The first objective of this work was to derive and apply new methodologies based on the inherent qualities of the pathway. We developed a new bacterial two-hybrid system that capitalizes on the folding quality control mechanism of the Tat pathway. One protein (prey) is fused to Tat-specific signal peptide. A second (bait) protein is produced as a fusion to a reporter that produces a "signal" (growth or enzymatic activity) only when the bait-reporter fusion binds to the prey and the resulting complex is exported into the periplasm via the Tat pathway. As a second biotechnological application of the Tat pathway, we developed a phage display system that allows the protein of interest to fold within the cytoplasm prior export and display onto phage particles. This is in contrast to the conventional phage display system, in which displayed protein folds in the periplasm. We took advantage of this new system to screen a library of 2 x 10⁶ of fluorescent GFP variants containing a hexameric peptide insertion for ligand binding. Despite the diversity of the hexamer, we were not able to isolate single GFP variants that would bind with specificity to various ligands. This highlights the difficulty in engineering GFP variants that can bind to other proteins while retaining the ability to fluoresce. The second aspect of this research was to examine mechanistic aspects of the Tat pathway. TatB and TatC are responsible for the recognition of Tat signal peptides. Here, we established the importance of TatC as the crucial component of the Tat pathway for the interaction with the hallmark twin-arginine motif within Tat signal peptide. Substitution of the RR dipeptide with a KK sequence completely abolishes export. In a genetic screen using a ssTorA(KK)-GFP-SsrA as a reporter. We identified several amino acid substitutions within TatC that allowed the alteration of the substrate specificity of the pathway as indicated by the impairment of indigenous Tat substrates. Finally, we analyzed the conformational dynamics of TatA using GFP fusions and by incorporation of the chemically reactive, non-canonical amino acid azidohomoalanine.
Colorimetric bisulfite sensing and thio-amine click and declick reactions with applications in polymer chemistry and biotechnology
(2017-08-07) Kolesnichenko, Igor Vladimir; Anslyn, Eric V., 1960-; Sessler, Jonathan L; Jones, Richard A; Hoffman, David W; Lynd, Nathaniel A
Sulfur dioxide and its derivative, bisulfite, are crucial components of food and wine because of their antioxidant and antibacterial properties. While these compounds, often referred to as “sulfites,” are important, their optimal concentration range is very narrow and exceeding it can cause adverse effects, ranging from altered taste to allergic reactions in consumers. The current internationally recognized methods used for sensing sulfites have drawbacks, such as lengthy procedures and low accuracy. Thus, the motivation of this research is to develop an efficient and more accurate technique for sensing sulfites. The initial approach was to couple the host-guest chemistry of cucurbiturils with the known reactivity of bisulfite with carbonyl compounds to cause a shift in λ [subscript max] of the complexes; however, a compound was found which could alone cause a color change upon reaction with bisulfite. Moreover, hydrogen peroxide was formed in the process and the current focus is to quantitatively measure the amount of peroxide generated to back-calculate the amount of bisulfite initially present. Separately, an important goal of organic chemistry has centered on the development of methods to assemble and disassemble molecular modules. The focus of this work is the development of a molecular tether to reversibly bind amines and thiols in aqueous conditions at neutral pH. Herein, are the details of reactions using a Meldrum’s acid-derived conjugate acceptor and its utility in peptide and polymer chemistries, which could greatly benefit from the introduction of reversible covalent bonding units. A current focus of pharmaceutical companies is the attachment of polyethylene glycol (PEG) groups to drugs and proteins to improve water solubility and increase hydrodynamic radius. However, high molecular weight PEG chains can bioaccumulate within the body. One focus of this work is the development of high molecular weight PEG chains that can be chemically triggered to break down into smaller units. In a separate field, a trend in the development of new bactericidal compounds is the use of antibacterial peptides that withstand decomposition through cyclization. Thus, another application of the aforementioned conjugate acceptor is the reversible cyclization of antibacterial peptides to both mask them from enzymes and modulate their activity.
Development of tools for single molecule peptide fluorosequencing
(2021-09-16) Hinson, Caroline Marie; Anslyn, Eric V., 1960-; Brodbelt, Jennifer; Schiavinato Eberlin, Livia; Keitz, Benjamin K; Lynd, Nathaniel; Marcotte, Edward
Fluorosequencing, technology that combines selective fluorophore labeling of amino acid residues, total-internal reflection fluorescence (TIRF) microscopy, and Edman degradation, is a novel technology that has been developed to sequence peptides. Unique fluorophores are covalently attached to amino acid residues and through TIRF microscopy are traced through Edman degradation cycles that remove one amino acid at a time. By determining which Edman cycles fluorescence is lost sequence information can be obtained. This has been shown to be a useful tool for determining the sequence of peptides in a highly parallel manner. As each molecule is individually monitored, this single molecule method is useful for the discovery of low abundance peptides and single cell proteomics. New tools are needed to improve sequencing and label new amino acid residues in selective fashion with fluorophores. Single molecule peptide fluorosequencing has unique requirements. Surfaces need to selectively attach peptides by their C-terminus but resist non-specifically bound fluorophores throughout an entire fluorosequencing experiment. Developing a surface that would be suitable for fluorosequencing is detailed in Chapter 2. Preparing peptides for fluorosequencing also has many challenges. Labeling amino acid residues with fluorophores requires multiple steps and purification that can erode the number of peptides in a sample with each step. This can be overcome with a solid support that captures peptides by their N-termini and enables chemical labeling without the loss of sample (Chapter 3). Additional labeling schemes, that are selective and high yield, are needed to expand the number of amino acid residues capable of being sequenced by fluorosequencing. Two-step labeling opens new possibilities for labeling of amino acids without fluorophore degradation. This is demonstrated in the two-step labeling of glutamic acid with dithiol-aldehyde labeling (Chapter 4). A fluorophore linker with a 1,3-dithiol is synthesized for the labeling of aldehydes, which was done in an efficient manner by deprotecting the aldehyde and labeling in one step. Another two-step labeling scheme was developed for labeling the locations of glycosylation on peptides. By exploiting the susceptibility of glycosylations to β-elimination over phosphorylation, glycosylations were selectively labeled in the presence of phosphorylations and fluorosequenced (Chapter 5).
Flanking Residues Are Central to DO11.10 T Cell Hybridoma Stimulation by Ovalbumin 323–339
(Public Library of Science, 2012-10-23) Roy, Benjamin M.; Zhukov, Dmitriy V.; Maynard, Jennifer A.
T cell activation requires formation of a tri-molecular interaction between a major histocompatibility complex (MHC), peptide, and T cell receptor. In a common model system, the ovalbumin epitope 323–339 binds the murine class II MHC, I-Ad, in at least three distinct registers. The DO11.10 T cell recognizes the least stable of these, as determined by peptide-MHC dissociation rates. Using exogenous peptides and peptide insertions into a carrier protein in combination with IL-2 secretion assays, we show that the alternate registers do not competitively inhibit display of the active register four. In contrast, this weakly binding register is stabilized by the presence of N-terminal flanking residues active in MHC binding. The DO11.10 hybridoma is sensitive to the presence of specific wild-type residues extending to at least the P-3 peptide position. Transfer of the P-4 to P-2 flanking residues to a hen egg lysozyme epitope also presented by I-Ad increases the activity of that epitope substantially. These results illustrate the inherent complexity in delineating the interaction of multiple registers based on traditional thermodynamic measurements and demonstrate the potential of flanking residue modification for increasing the activity of weakly bound epitopes. The latter technique represents an alternative to substitution of anchor residues within a weakly bound register, which we show can significantly decrease the activity of the epitope to a responding T cell.
FRET peptidyl sensors for the detection of metal ions
(2007) White, Brianna Rose, 1981-; Holcombe, James A.
This research focuses on developing selective FRET peptidyl metal ion sensors as a portable and less costly alterative to traditional atomic spectrometric techniques. Initially, a selective sensor for Cu²⁺ was developed that consisted of glycine and aspartic acid residues and the FRET pair tryptophan (donor) and dansyl (acceptor). Aspartic acid's affinity for hard acid metals and Cu²⁺'s preference for square planar coordination was used as the basis of design. Although the sensor was designed to utilize the signal enhancement capabilities of FRET, quenching of both fluorophores occurred and proved to be the most sensitive means of quantifying Cu²⁺ binding. Nonetheless, the sensor provided a selective and sensitive response to Cu²⁺ at pH 7.0. Another FRET peptide metal ion sensor was designed with the help of a biological starting point, the mercury binding protein MerP. A sensitive FRET enhancement or "turn on" response was observed for Hg²⁺, as well as Zn²⁺, Cd²⁺ and Ag²⁺ in pH 7.0 solution. While a selective response for only Hg²⁺ was the ultimate goal of this study, this sensor is still an improvement over current systems which utilize a quenching mechanism for Hg²⁺ detection. While the previous studies investigated these sensors in aqueous solutions, the end goal was to devise a sensor based on an immobilized peptide chelator with FRET capabilities. To this end, immobilized, fluorophore labeled peptide studies were then conducted on Tentagel resin using a visible region FRET pair. A flow injection fluorescence analysis system using the immobilized fluorophore labeled peptide as the ion exchange material was also designed, allowing for the efficient analysis of fluorescence solutions. In addition to the work conducted with FRET sensors, studies were also conducted using magnetic [gamma]-Fe₂O₃ nanoparticles with PLCys immobilized onto the surface. The [gamma]-Fe₂O₃ nanoparticles are ideal supports since they can be magnetically collected and have a very large surface area to mass ratio. Finally, a method was developed to quantitatively screen metals bound to single Tentagel beads with immobilized peptides using ETV-ICP-MS. This method is an improvement over existing methods because it is nondestructive and simultaneously provides the absolute content of all metals bound.
Integration and validation of mass spectrometry proteomics data sets
(2008-05) Prince, John Theodore, 1976-; Marcotte, Edward M.
Mass spectrometry (MS) has been a key player in biological investigation for some time and is the instrument of choice for high throughput proteomics. However, the generation of large, inherently rich, proteomics data sets has far outpaced our ability to utilize them to produce biological knowledge. The ultimate utility of MS proteomics is closely tied to our ability to interpret, integrate and validate this voluminous data. By way of introduction, I discuss the creation of the Open Proteomics Database, which aims to increase publicly available data and to encourage broader contribution from the statistical and bioinformatic communities. Next, I detail research efforts in the integration of mass spectrometry data sets to increase the number of quantifiable peptides. Comparing peptide quantities between experiments (or subsequent chromatographic fractions) in large numbers requires the chromatographic alignment of MS signals, a challenging problem. We use Dynamic Time Warping (DTW) and a bijective (one-to-one) interpolant to create a smooth warp function amenable to multiple alignment. We test a wide variety of alignment scenarios coupled with high confidence, overlapping peptide identifications to optimize and compare alignment parameters. We determine an optimal spectral similarity function, show the importance of penalizing gaps in the alignment path, and demonstrate the utility of our algorithm for multiple alignments. Then, we introduce a method to independently validate large scale proteomics data sets. We use known biases in sample constitution including amino acid content, transmembrane sequence content, and protein abundance to estimate peptide false identification rates (FIRs) in what we term sample bias validation (SBV). We use SBV to compare the false identification rate accuracy (FIRA) and recall capabilities of widely used techniques for error estimation in MS based proteomics. Finally, we describe the open source package mspire (mass spectrometry proteomics in Ruby). Mspire offers unified interfaces for working with a variety of file formats across the analytical pipeline, much needed converters between key formats, and tools for FIR determination. The package eases the burden of working with MS proteomics data, reducing the barrier of entry to developers and offering useful tools to analysts of MS proteomics data.
Isolation and Characterization of CvIV4: A Pain Inducing α- Scorpion Toxin
(Public Library of Science, 2011-08-24) Rowe, Ashlee H.; Xiao, Yucheng; Scales, Joseph; Linse, Klaus D.; Rowe, Matthew P.; Cummins, Theodore R.; Zakon, Harold
Background -- Among scorpion species, the Buthidae produce the most deadly and painful venoms. However, little is known regarding the venom components that cause pain and their mechanism of action. Using a paw-licking assay (Mus musculus), this study compared the pain-inducing capabilities of venoms from two species of New World scorpion (Centruroides vittatus, C. exilicauda) belonging to the neurotoxin-producing family Buthidae with one species of non-neurotoxin producing scorpion (Vaejovis spinigerus) in the family Vaejovidae. A pain-inducing α-toxin (CvIV4) was isolated from the venom of C. vittatus and tested on five Na+ channel isoforms. Principal Findings -- C. vittatus and C. exilicauda venoms produced significantly more paw licking in Mus than V. spinigerus venom. CvIV4 produced paw licking in Mus equivalent to the effects of whole venom. CvIV4 slowed the fast inactivation of Nav1.7, a Na+ channel expressed in peripheral pain-pathway neurons (nociceptors), but did not affect the Nav1.8-based sodium currents of these neurons. CvIV4 also slowed the fast inactivation of Nav1.2, Nav1.3 and Nav1.4. The effects of CvIV4 are similar to Old World α-toxins that target Nav1.7 (AahII, BmK MI, LqhIII, OD1), however the primary structure of CvIV4 is not similar to these toxins. Mutant Nav1.7 channels (D1586A and E1589Q, DIV S3–S4 linker) reduced but did not abolish the effects of CvIV4. Conclusions -- This study: 1) agrees with anecdotal evidence suggesting that buthid venom is significantly more painful than non-neurotoxic venom; 2) demonstrates that New World buthids inflict painful stings via toxins that modulate Na+ channels expressed in nociceptors; 3) reveals that Old and New World buthids employ similar mechanisms to produce pain. Old and New World α-toxins that target Nav1.7 have diverged in sequence, but the activity of these toxins is similar. Pain-inducing toxins may have evolved in a common ancestor. Alternatively, these toxins may be the product of convergent evolution.
A Model of a MAPK•Substrate Complex in an Active Conformation: A Computational and Experimental Approach
(Public Library of Science, 2011-04-11) Lee, Sunbae; Warthaka, Mangalika; Yan, Chunli; Kaoud, Tamer S.; Piserchio, Andrea; Ghose, Ranajeet; Ren, Pengyu; Dalby, Kevin N.
The mechanisms by which MAP kinases recognize and phosphorylate substrates are not completely understood. Efforts to understand the mechanisms have been compromised by the lack of MAPK-substrate structures. While MAPK-substrate docking is well established as a viable mechanism for bringing MAPKs and substrates into close proximity the molecular details of how such docking promotes phosphorylation is an unresolved issue. In the present study computer modeling approaches, with restraints derived from experimentally known interactions, were used to predict how the N-terminus of Ets-1 associates with ERK2. Interestingly, the N-terminus does not contain a consensus-docking site ((R/K)2-3-X2-6-ΦA-X-ΦB, where Φ is aliphatic hydrophobic) for ERK2. The modeling predicts that the N-terminus of Ets-1 makes important contributions to the stabilization of the complex, but remains largely disordered. The computer-generated model was used to guide mutagenesis experiments, which support the notion that Leu-11 and possibly Ile-13 and Ile-14 of Ets-1 1-138 (Ets) make contributions through binding to the hydrophobic groove of the ERK2 D-recruiting site (DRS). Based on the modeling, a consensus-docking site was introduced through the introduction of an arginine at residue 7, to give the consensus 7RK-X2-ΦA-X-ΦB13. This results in a 2-fold increase in kcat/Kmfor the phosphorylation of Ets by ERK2. Similarly, the substitution of the N-terminus for two different consensus docking sites derived from Elk-1 and MKK1 also improves kcat/Km by two-fold compared to Ets. Disruption of the N-terminal docking through deletion of residues 1-23 ofEts results in a 14-fold decrease in kcat/Km, with little apparent change in kcat. A peptide that binds to the DRS of ERK2 affects Km, but not kcat. Our kinetic analysis suggests that the unstructured N-terminus provides 10-fold uniform stabilization of the ground state ERK2•Ets•MgATP complex and intermediates of the enzymatic reaction.
Mre11 Assembles Linear DNA Fragments into DNA Damage Signaling Complexes
(Public Library of Science, 2004-05-11) Costanzo, Vincenzo; Paull, Tanya; Gottesman, Max; Gautier, Jean
Mre11/Rad50/Nbs1 complex (MRN) is essential to suppress the generation of double-strand breaks (DSBs) during DNA replication. MRN also plays a role in the response to DSBs created by DNA damage. Hypomorphic mutations in Mre11 (which causes an ataxia-telangiectasia-like disease [ATLD]) and mutations in the ataxia-telangiectasia-mutated (ATM) gene lead to defects in handling damaged DNA and to similar clinical and cellular phenotypes. Using Xenopus egg extracts, we have designed a simple assay to define the biochemistry of Mre11. MRN is required for efficient activation of the DNA damage response induced by DSBs. We isolated a high molecular weight DNA damage signaling complex that includes MRN, damaged DNA molecules, and activated ATM. Complex formation is partially dependent upon Zn2+ and requires an intact Mre11 C-terminal domain that is deleted in some ATLD patients. The ATLD truncation can still perform the role of Mre11 during replication. Our work demonstrates the role of Mre11 in assembling DNA damage signaling centers that are reminiscent of irradiation-induced foci. It also provides a molecular explanation for the similarities between ataxia-telangiectasia (A-T) and ATLD.
Mutant peptides and proteins : a molecular inquiry into biological form and function
(1995-12) Spaller, Mark Robert; Not available
Nanoimprint lithography based fabrication of size and shape-specific, enzymatically-triggered nanoparticles for drug delivery applications
(2008-05) Glangchai, Luz Cristal Sanchez, 1977-; Roy, Krishnendu; Shi, Li, Ph. D.
Our ability to precisely manipulate size, shape, and composition of nanoscale carriers is essential for controlling their in-vivo transport, biodistribution, and drug release mechanism. Shape-specific, "smart" nanoparticles that deliver drugs or imaging agents to target tissues primarily in response to disease-specific or physiological signals could significantly improve therapeutic care of complex diseases. Current methods in nanoparticle synthesis do not allow such simultaneous control over particle size, shape, and environmentally-triggered drug release, especially at the sub-100 nm range. In this dissertation, we discuss the development of high-throughput nanofabrication techniques using synthetic and biological macromers (peptides) to produce highly monodisperse nanoparticles, as well as enzymatically-triggered nanoparticles, of precise sizes and shapes. We evaluated thermal nanoimprint lithography (ThNIL) and step and flash imprint lithography (SFIL) as two possible fabrication techniques. We successfully employed ThNIL and SFIL for fabricating nanoparticles and have extensively characterized the SFIL fabrication process, as well as the properties of the imprinted biopolymers. Particles as small as 50 nm were fabricated on silicon wafers and harvested directly into aqueous buffer using a biocompatible, one-step release technique. These methods provide a novel way to fabricate biocompatible nanoparticles with precise size and geometry. Furthermore, we developed an enzyme-degradable material system and demonstrated successful encapsulation and enzyme-triggered release of antibodies and nucleic acids from these imprinted nanoparticles; thus providing a potential means for disease-controlled delivery of biomolecules. Finally, we evaluated the bioactivity of the encapsulated therapeutics in-vitro. The development of the SFIL method for fabrication of biocompatible nanocarriers has great potential in the drug delivery field for its ability to create monodisperse particles of pre-designed geometry and size, and to incorporate stimulus-responsive release mechanisms. This research provides the potential to broaden the study of how particle size and shape affect the biodistribution of drugs within the body.
Single scaffold antibody libraries created with high rates of mutagenesis or diversity focused for peptide recognition
(2007-12) Cobaugh, Christian Wessel, 1971-; Iverson, Brent L.; Georgiou, George
This dissertation describes several strategies used to create diversity in non-immune antibody libraries. Two of the strategies were used to create two separate peptide focused libraries. Both of these strategies used to create these antigen-class focused libraries used a single scaffold antibody gene that contained diversity only in the variable heavy region. The scaffold antibody gene one of the libraries, the M:anti-pep library, was chosen based on hypervariable loop canonical structures that are characteristic of other anti-peptide antibodies. Additionally, all of the contact residues of this antibody are commonly used contact residues in other anti-peptide antibodies. These positions and others were varied to incorporate the natural diversity of other anti-peptide antibodies. The second library, the Hu:anti-pep, is based on a widely used, unique combination of human germline antibody segments that express well in bacterial expression. Positions were chosen for variation based on their usage as contact residues in both anti-peptide and anti-protein antibodies. The diversity was less focused than with the M:anti-pep library, incorporating all 20 amino acids at "high usage" positions and only four amino acids at "low usage" positions. Both libraries were validated by phage display selections against the peptide angiotensin (AT) and neuropeptide Y (NPY). The M:anti-pep library yielded specific antibodies to both peptides with dissociation constants as low as 14 nM against AT and 18 nM against NPY. The Hu:anti-pep library yielded specific clones with higher dissociation constants: 49 nM against NPY and 11 [mu]M against AT. The final strategy used to introduce diversity is widely used for affinity maturation of low affinity, previously selected antibodies. Extremely high rates of mutagenesis (2.2% of the gene to 2.7%) were used to create two libraries of the anti-digoxin antibody 26-10. The libraries had been screened by others in an attempt to examine the effects of highrates of mutagenesis on the directed evolution of an antibody. A total of 91 isolated clones from both libraries were sequenced. Several consensus mutations were identified near the CDRH3 in the isolated clones, indicating that they had a positive, selectable effect. This study confirmed that high-error rate antibody libraries contain more active clones than expected. Combinations of the selected consensus mutations from these libraries provide moderate enhancements to the kinetics and expression of the wild-type antibody in a non-synergistic manner.
Synthesis and characterization of short-chain peptides for use in metal remediation and preconcentration
(2006) Stair, Jacqueline Leslie; Holcombe, James A.
Designing materials for metal remediation and preconcentration based on naturally occurring metal binding proteins has become of growing interest due to their inherent selective and strong binding, ease of synthesis and available amino acid building blocks, and environmental innocuity. One approach is through the use of immobilized synthetic biohomopolymers which can provide the selectivity based on the amino acid side chain moiety with strong binding, easy on-demand release, and reusability. An attempt to increase metal binding selectivity of these biohomopolymers was done though cross-linking at specific locations as to effectively “lock” in place the preferential binding cavity for a particular metal. The cross-linking of these materials resulted in decreased metal capacities with little to no increases in the targeted metal selectivity. This was likely due to the loss of bound metal during cross-linking and to a lack of rigidity in the overall cross-linked polymer. Short composite peptides synthesized on a commercially available resin, TentaGel were also examined as a means to increase metal selectivity. These peptides showed surprisingly high metal binding capacities, strong binding, and residue per metal binding ratios which were an order of magnitude better than results previously reported for longer chain poly-amino acids (50 – 70 residues) attached to porous glass supports. Metal binding selectivity’s were altered by changing only one amino acid and metal release under acidic conditions was surprisingly rapid for these shorter peptides. As a result of these findings, the metal binding and conformational changes between TentaGel immobilized short and long peptide chains during metal binding and release were monitored using Raman microscopy. These results indicated that metal release occurred via conformational changes in addition to proton displacement. Lastly, a method to screen multi-metal binding capabilities of combinatorial peptide libraries was developed using electrothermal vaporization inductively coupled plasma mass spectrometry (ETV-ICP-MS). With the exception of metals that are bound tightly to the peptide, acid stripping of the metals in a single bead into a small volume appears as a viable quantitative analytical approach when using this method with instrumental precisions of better than ±10% for most metals when larger polymer beads (~250 µm) were employed.
Synthesis and evaluation of conformationally constrained peptide replacements and studies toward the total synthesis of kidamycin
(2004) Plake, Hilary Ruth; Martin, Stephen F.
A series of conformationally constrained pseudopeptides derivative of the tripeptide pYVN were designed and synthesized. The conformationally restricted compounds contained either trans- or cis-cyclopropanes as replacements to enforce locally extended and reverse turn peptide conformations, respectively. In addition, the proper flexible control molecules were prepared. All compounds were evaluated for the ability to bind to the Grb2-SH2 domain in order to determine the energetic consequences of introducing a conformational constraint into peptide ligands. No difference in the ∆Gbinding between the trans-cyclopropane and its control partner was observed. Surprisingly, there was an entropic disadvantage when comparing the binding energetics of the constrained and flexible pseudopeptides. Therefore, the introduction of the cyclopropane constraint was associated with an entropic disadvantage in the system, which is the opposite of conventional wisdom. An X-ray crystal structure of the trans- cyclopropane containing ligand bound to the Grb2-SH2 domain was obtained and vii discussed. On the other hand, cis-cyclopropane containing pseudopeptides do not seem to enforce the desired turn conformation of the ligand. A method to allow access to unsymmetrical C-aryl glycoside natural products was developed through employing a disposal tether to enforce the desired regioselectivity in a [4+2] cycloaddition between benzyne and glycosyl-substituted furan. Application of this novel strategy toward the synthesis of kidamycin is discussed. Additional synthetic routes, including utilizing Suzuki’s O → C glycoside rearrangement are also provided. Studies toward the synthesis of sugar ring E and F are illustrated.
Towards peptide-binding peptides
(2001-08) Zhang, Zhiwen; Anslyn, Eric V., 1960-; Kodadek, Thomas J.
Peptide-binding peptides can be a very powerful research tool. A novel methodology, based on the mechanism of bacteriophage l switch in E. coli and combinatorial screening, has been developed to isolate peptides that bind another target peptide in vivo. Two pairs of interacting peptides have been isolated and characterized. One of the potential applications of such peptide-binding peptides is to be utilized as protein purification tags. Another novel aspect of this research is that a candidate peptide is able to inhibit an enzyme-catalyzed protein hydrolysis by binding specifically to a peptide sequence on the substrate which is recognized by the protease. In other words, a novel concept of substrate-directed enzyme inhibitors has been developed.