Native Tryptophan Research Articles

Enhancing Gastrodin Production in Yarrowia lipolytica by Metabolic Engineering.

Gastrodin, 4-hydroxybenzyl alcohol-4-O-β-D-glucopyranoside, has been widely used in the treatment of neurogenic and cardiovascular diseases. Currently, gastrodin biosynthesis is being achieved in model microorganisms. However, the production levels are insufficient for industrial applications. In this study, we successfully engineered a Yarrowia lipolytica strain to overproduce gastrodin through metabolic engineering. Initially, the engineered strain expressing the heterologous gastrodin biosynthetic pathway, which comprises chorismate lyase, carboxylic acid reductase, phosphopantetheinyl transferase, endogenous alcohol dehydrogenases, and a UDP-glucosyltransferase, produced 1.05 g/L gastrodin from glucose in a shaking flask. Then, the production was further enhanced to 6.68 g/L with a productivity of 2.23 g/L/day by overexpressing the key node DAHP synthases of the shikimate pathway and alleviating the native tryptophan and phenylalanine biosynthetic pathways. Finally, the best strain, Gd07, produced 13.22 g/L gastrodin in a 5 L fermenter. This represents the highest reported production of gastrodin in an engineered microorganism to date, marking the first successful de novo production of gastrodin using Y. lipolytica.

Temporal Resolution of Activity-Related Solvation Dynamics in the TIM Barrel Enzyme Murine Adenosine Deaminase.

Murine adenosine deaminase (mADA) is a prototypic system for studying the thermal activation of active site chemistry within the TIM barrel family of enzyme reactions. Previous temperature-dependent hydrogen deuterium exchange studies under various conditions have identified interconnected thermal networks for heat transfer from opposing protein-solvent interfaces to active site residues in mADA. One of these interfaces contains a solvent exposed helix-loop-helix moiety that presents the hydrophobic face of its long α-helix to the backside of bound substrate. Herein we pursue the time and temperature dependence of solvation dynamics at the surface of mADA, for comparison to established kinetic parameters that represent active site chemistry. We first created a modified protein devoid of native tryptophans with close to native kinetic behavior. Single site-specific tryptophan mutants were back inserted into each of the four positions where native tryptophans reside. Measurements of nanosecond fluorescence relaxation lifetimes and Stokes shift decays, that reflect time dependent environmental reoroganization around the photo-excited state of Trp*, display minimal temperature dependences. These regions serve as controls for the behavior of a new single tryptophan inserted into a solvent exposed region near the helix-loop-helix moiety located behind the bound substrate, Lys54Trp. This installed Trp displays a significantly elevated value for ; further, when Phe61 within the long helix positioned behind bound substrate is replaced by a series of aliphatic hydrophobic side chains, the trends in mirror the earlier reported impact of the same series of function-altering hydrophobic side chains on the activation energy of catalysis, .The reported experimental findings implicate a solvent initiated and rapid (>ns) protein restructuring that contributes to the enthalpic activation barrier to catalysis in mADA.

Calcium dependence of both lobes of calmodulin is involved in binding to a cytoplasmic domain of SK channels.

KCa2.1-3 Ca2+-activated K+-channels (SK) require calmodulin to gate in response to cellular Ca2+. A model for SK gating proposes that the N-terminal domain (N-lobe) of calmodulin is required for activation, but an immobile C-terminal domain (C-lobe) has constitutive, Ca2+-independent binding. Although structures support a domain-driven hypothesis of SK gate activation by calmodulin, only a partial understanding is possible without measuring both channel activity and protein binding. We measured SK2 (KCa2.2) activity using inside-out patch recordings. Currents from calmodulin-disrupted SK2 channels can be restored with exogenously applied calmodulin. We find that SK2 activity only approaches full activation with full-length calmodulin with both an N- and a C-lobe. We measured calmodulin binding to a C-terminal SK peptide (SKp) using both composition-gradient multi-angle light-scattering and tryptophan emission spectra. Isolated lobes bind to SKp with high affinity, but isolated lobes do not rescue SK2 activity. Consistent with earlier models, N-lobe binding to SKp is stronger in Ca2+, and C-lobe-binding affinity is strong independent of Ca2+. However, a native tryptophan in SKp is sensitive to Ca2+ binding to both the N- and C-lobes of calmodulin at Ca2+ concentrations that activate SK2, demonstrating that the C-lobe interaction with SKp changes with Ca2+. Our peptide-binding data and electrophysiology show that SK gating models need deeper scrutiny. We suggest that the Ca2+-dependent associations of both lobes of calmodulin to SKp are crucial events during gating. Additional investigations are necessary to complete a mechanistic gating model consistent with binding, physiology, and structure.

Bioinspired Hybrid Fluorescent Ligands for the FK1 Domain of FKBP52.

The protein FKBP52 is a steroid hormone receptor coactivator likely involved in neurodegenerative disease. A series of small, water-soluble, bioinspired, pseudopeptidic fluorescent ligands for the FK1 domain of this protein are described. The design is such that engulfing of the ligand in the pocket of this domain is accompanied by hydrogen-bonding of the dansyl chromophore which functions as both an integral part of the ligand and a fluorescent reporter. Binding is concomitant with a significant wavelength shift and an enhancement of the ligand fluorescence signal. Excitation of FK1 domain native tryptophan residues in the presence of bound ligand results in Förster resonance energy transfer. Variation of key ligand residues within the short sequence was undertaken, and the interaction of the resulting library with the protein was measured by techniques including isothermal calorimetry analysis, fluorescence, and FRET quenching, and a range of Kd values were determined. Cocrystallization of a protein ligand complex at 2.30 Å resolution provided detailed information at the atomic scale, while also providing insight into native substrate binding.

Site-Specific Tryptophan Labels Reveal Local Microsecond-Millisecond Motions of Dihydrofolate Reductase.

Many enzymes are known to change conformations during their catalytic cycle, but the role of these protein motions is not well understood. Escherichia coli dihydrofolate reductase (DHFR) is a small, flexible enzyme that is often used as a model system for understanding enzyme dynamics. Recently, native tryptophan fluorescence was used as a probe to study micro- to millisecond dynamics of DHFR. Yet, because DHFR has five native tryptophans, the origin of the observed conformational changes could not be assigned to a specific region within the enzyme. Here, we use DHFR mutants, each with a single tryptophan as a probe for temperature jump fluorescence spectroscopy, to further inform our understanding of DHFR dynamics. The equilibrium tryptophan fluorescence of the mutants shows that each tryptophan is in a different environment and that wild-type DHFR fluorescence is not a simple summation of all the individual tryptophan fluorescence signatures due to tryptophan–tryptophan interactions. Additionally, each mutant exhibits a two-phase relaxation profile corresponding to ligand association/dissociation convolved with associated conformational changes and a slow conformational change that is independent of ligand association and dissociation, similar to the wild-type enzyme. However, the relaxation rate of the slow phase depends on the location of the tryptophan within the enzyme, supporting the conclusion that the individual tryptophan fluorescence dynamics do not originate from a single collective motion, but instead report on local motions throughout the enzyme.

Dark, Ultra-Dark and Ultra-Bright Nanodiscs for membrane protein investigations

We describe the construction, expression and purification of three new membrane scaffold proteins (MSP) for use in assembling Nanodiscs. These new MSPs have a variety of luminescent properties for use in combination with several analytical methods. “Dark” MSP has no tryptophan residues, “Ultra-Dark” replaces both tryptophan and tyrosine with non-fluorescent side chains, and “Ultra-Bright” adds additional tryptophans to the parent membrane scaffold protein to provide a dramatic increase in native tryptophan fluorescence. All MSPs were used to successfully assemble Nanodiscs nominally 10 nm in diameter, and the resultant bilayer structure was characterized. An example of the usefulness of these new scaffold proteins is provided.

Replacing the eleven native tryptophans by directed evolution produces an active P-glycoprotein with site-specific, non-conservative substitutions

P-glycoprotein (Pgp) pumps an array of hydrophobic compounds out of cells, and has major roles in drug pharmacokinetics and cancer multidrug resistance. Yet, polyspecific drug binding and ATP hydrolysis-driven drug export in Pgp are poorly understood. Fluorescence spectroscopy using tryptophans (Trp) inserted at strategic positions is an important tool to study ligand binding. In Pgp, this method will require removal of 11 endogenous Trps, including highly conserved Trps that may be important for function, protein-lipid interactions, and/or protein stability. Here, we developed a directed evolutionary approach to first replace all eight transmembrane Trps and select for transport-active mutants in Saccharomyces cerevisiae. Surprisingly, many Trp positions contained non-conservative substitutions that supported in vivo activity, and were preferred over aromatic amino acids. The most active construct, W(3Cyto), served for directed evolution of the three cytoplasmic Trps, where two positions revealed strong functional bias towards tyrosine. W(3Cyto) and Trp-less Pgp retained wild-type-like protein expression, localization and transport function, and purified proteins retained drug stimulation of ATP hydrolysis and drug binding affinities. The data indicate preferred Trp substitutions specific to the local context, often dictated by protein structural requirements and/or membrane lipid interactions, and these new insights will offer guidance for membrane protein engineering.

Both Lobes of Calmodulin Bound to KCa2.2 Respond to Ca2+

An increase in intracellular Ca2+ opens KCa2.2 Ca2+-activated K+-channels (SK) to conduct K+ down its electrochemical gradient. SK activation requires bound calmodulin proteins to decode dynamic Ca2+ fluctuations in a cell. Bilobal calmodulin has four Ca2+ binding sites. A common model proposes that just the N-terminal domain (N-lobe) of calmodulin binds Ca2+ to open SK, while an immobile C-terminal domain (C-lobe) has constitutive, Ca2+-independent binding. Such a mechanism is compatible with x-ray and EM structures of intact SK channels and fragments. While molecular structures provide snapshots of the channels in different conformations with and without calcium, gating is a dynamic process involving several different conformations, calcium binding to calmodulin, interactions between calmodulin and the channel and channel opening. We measured SK activity using inside-out patch recordings from “calmodulin-disrupted” SK channels where calcium-dependent activation can be restored by exogenously applied full-length calmodulin. Isolated N and C lobes have little ability to activate SK. We measured calmodulin binding to a C-terminal SK2 peptide (SKp) using both composition-gradient multi-angle light scattering and tryptophan emission spectra. While they do not rescue activation, isolated lobes do bind to a SK cytoplasmic domain with high affinity. Consistent with earlier models, N-lobe binding to SKp is stronger in Ca2+, and C-lobe binding affinity is strong regardless of Ca2+. However, a native tryptophan in SKp is sensitive to Ca2+ binding to either the N- or C-lobe of calmodulin at Ca2+ concentrations that activate SK. Our results show that the calmodulin C-lobe bound to SK is Ca2+ sensitive and that both lobes of calmodulin bind SK in Ca2+-dependent states governed by the different properties of each lobe.

Direct Visualization of Platelet Integrins Using ANTI-Transmembrane Domain Peptides Containing a BLUE Fluorescent Amino Acid

Antibodies containing a fluorescent label or recombinant proteins containing a fluorescent reporter are commonly used to visualize proteins in situ. However, antibodies and bulky reporters can perturb protein structure and function. To overcome this problem, we have designed a minimally perturbing blue fluorescent unnatural amino acid that enables the direct imaging of intrinsically-fluorescent proteins with their interaction partners. Specifically, we used a nitrile-derivatized tryptophan, 4-cyanotryptophan (4CNTrp), which differs by only two atoms from native tryptophan. 4CNTrp has unique photophysical properties in the visible blue region: an absorption maximum at ~325 nm, an emission maximum at ~420 nm, a large fluorescence quantum yield (>0.8 in aqueous solution), a long fluorescence lifetime (ca. 13 ns), and good photostability (Hilaire et al. PNAS 2017; 114: 6005-09). Based on these properties, 4CNTrp-labeling was recently used to visualize the binding of a model peptide pHLIP (pH-(Low) Insertion Peptide) to cell membranes via wide-field fluorescence microscopy. 4CNTrp is also a viable FRET donor for acceptor pairs in the green visible region. 4CNTrp can be prepared by a simple, high-yielding cost-effective synthetic route and because it does not inhibit bacterial growth, it may be practical for cellular applications (Zhang et al. Chem. Comm. 2019; 55: 5095-98). Here, we used 4CNTrp-labeled peptides to directly image integrins on cell surfaces. Previously, we described the synthesis of peptides, called CHAMP peptides for Computed Helical Anti-Membrane Protein, that target the transmembrane (TM) domains of integrins in a sequence-specific manner (Yin et al. Science 2007; 315: 1817-22). Thus, the CHAMP peptides anti-αIIb and anti-αv specifically bind to the TM domains of αIIb and αv in platelet membranes, causing separation of the αIIbβ3 and αvβ3 TM domains and αIIbβ3 and αvβ3 activation. Anti-αIIb also caused αIIbβ3 clustering that was visualized using the β3-specific monoclonal antibody SSA6 labeled with Alexa Fluor 488 but only after the platelets were fixed with paraformaldehyde to prevent antibody-induced clustering. To enable the direct visualization of αIIβ3, αvβ3, and α2β1 on cell surfaces, we used solid-phase peptide synthesis to generate CHAMP peptides labeled with 4CNTrp at their N-termini (CN-αIIb, CN-αv, CN-β1). We found that 4CNTrp labeling did not perturb the ability of the CHAMP peptides to bind to integrins and to cause specific integrin activation. Thus, CN-αIIb caused αIIbβ3-dependent platelet aggregation, CN-αv caused the αvβ3-dependent platelet adhesion to osteopontin, and CN-α1 caused α2β1-mediated platelet adhesion to collagen. We then used high resolution wide-field deconvolution microscopy to image 4CNTrp-containing integrins on the surface of platelets, HEL cells, and transfected CHO cells. Compared to 4CNTrp-containing random peptides that uniformly decorate cell surfaces, the CN-labeled CHAMP peptides were present as a limited number of discrete foci on the cell surface. To confirm that these foci represented CN-peptide containing integrins, we co-stained the cells with Alexa Fluor 488-labeled integrin-specific monoclonal antibodies and found that CN-peptide and antibody fluorescence coincided. Thus, these studies demonstrate the specific and direct imaging of integrins embedded in cell membranes in their activated state using 4CNTrp-labeled anti-integrin TM peptides. Because 4CNTrp can readily be incorporated into proteins and peptides with little if any structural perturbation, 4CNTrp-labeling provides a facile way to directly monitor protein behavior and protein-protein interactions in cellular environments. Disclosures No relevant conflicts of interest to declare.

Characterization of the Assembly Dynamics of Streptococcus Pneumoniae FtsZ using Intrinsic Tryptophan Fluorescence

FtsZ is a prokaryotic cytoskeletal protein, which assembles into the Z‐ring at the division site and orchestrates the cell division process in bacteria. FtsZ has GTPase activity and self‐assembles to form polymers. We investigated the assembly characteristics of FtsZ from the pathogenic organism, Streptococcus pneumoniae. The critical concentration for FtsZ polymerization was found to be 2.4 ± 0.1 and 2.2 ± 0.2 μM measured using sedimentation assay and GTPase assay, respectively. The results indicated that the GTPase activity and assembly are closely linked to each other. We identified that FtsZ from Streptococcus species contains two native tryptophan (Trp) residues unique to this genus. Interestingly, we observed that during polymerization, the tryptophan fluorescence of FtsZ increases showing that the assembly of FtsZ can be monitored using the native tryptophan fluorescence. Using tryptophan fluorescence along with light scattering, sedimentation assay and electron microscopy we examined the effects of divalent cations, calcium and magnesium, on FtsZ assembly. Divalent magnesium enhanced the tryptophan fluorescence change during FtsZ assembly showing that it promoted the longitudinal assembly of FtsZ. Divalent calcium induced the bundling of FtsZ filaments thereby affecting the lateral interactions of FtsZ polymers but did not enhance the tryptophan fluorescence, showing the sensitivity of tryptophan fluorescence to longitudinal assembly. Next, we sought to investigate the contribution of individual tryptophan residues to fluorescence of FtsZ and use them for understanding the assembly of FtsZ. A homology model for FtsZ from Streptococcus pneumoniae showed that the two tryptophan residues were present at the C‐terminal of the protein – one at the 298th position of FtsZ near the T7 loop and the other at 378th position in the C‐terminal tail region. We used site‐directed mutagenesis to introduce point mutations to FtsZ at these positions to mutate individual tryptophan residues to cysteine or phenylalanine residues to create W294C/F and W378C/F mutant proteins. W378C/F mutants showed a similar change in tryptophan fluorescence during polymerization as of wild‐type, while W294C/F mutants did not show any change. This indicated that the Trp at the 294th position is responsible for the change in fluorescence during polymerization. This further suggested that the environment around 294W changes during the polymerization while the local environment around the 378W does not change. The two tryptophan residues in the protein allow us to use the intrinsic tryptophan fluorescence to gain new insights into FtsZ assembly. Further, it would be interesting to explore the use of these mutant proteins in following the domain movements and conformational changes occurring during the assembly and disassembly of FtsZ.Support or Funding InformationThe work is supported by a grant from Department of Science and Technology (DST, India) to D.P.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Polyallylamine hydrochloride coating enhances the fluorescence emission of Human Serum Albumin encapsulated gold nanoclusters

Protein encapsulated gold nanoclusters have received much attention due to the possibility of using them as a non-toxic fluorescent probe or marker for biomedical applications, however one major disadvantage currently is their low brightness and quantum yield in comparison to currently used fluorescent markers. A method of increasing the fluorescence emission of Human Serum Albumin (HSA) encapsulated gold nanoclusters (AuNCs) via a Polyallylamide hydrochloride (PAH) coating is described. PAH molecules with a molecular weight of ~17,500 Da were found to enhance the fluorescence emission of HSA-AuNCs by 3-fold when the protein/polymer concentration ratio is 2:1 in solution. Interestingly, the fluorescence lifetime of the AuNCs was found to decrease while the native tryptophan (TRP) fluorescence lifetime also decreased during the fluorescence emission intensity enhancement caused by the PAH binding. Coinciding with the decrease in fluorescence lifetime, the zeta potential of the system was observed to be zero during maximum fluorescence intensity enhancement, causing the formation of large aggregates. These results suggest that PAH binds to the HSA-AuNCs acting as a linker; causing aggregation and rigidification, which results in a decrease in separation between native TRP of HSA and AuNCs; improving Förster Resonance Energy Transfer (FRET) and increasing the fluorescence emission intensity. These findings are critical to the development of brighter protein encapsulated AuNCs.

Improved protein-crystal identification by using 2,2,2-trichloroethanol as a fluorescence enhancer

The identification of initial lead conditions for successful protein crystallization is crucial for structural studies using X-ray crystallography. In order to reduce the number of false-negative conditions, an emerging number of fluorescence-based methods have been developed which allow more efficient identification of protein crystals and help to distinguish them from salt crystals. Detection of the native tryptophan fluorescence of protein crystals is one of the most widely used methods. However, this method can fail owing to the properties of the crystallized protein or the chemical composition of the crystallization trials. Here, a simple, fast and cost-efficient method employing 2,2,2-trichloroethanol (TCE) has been developed. It can be performed with a standard UV-light microscope and can be applied to cases in which detection of native tryptophan fluorescence fails. In four test cases this method had no effect on the diffraction properties of the crystals and no structural changes were observed. Further evidence is provided that TCE can be added to crystallization trials during their preparation, making this method compatible with high-throughput approaches.

Painting argyrins blue: Negishi cross-coupling for synthesis of deep-blue tryptophan analogue β-(1-azulenyl)-l alanine and its incorporation into argyrin C

The argyrins are a family of non-ribosomal peptides that exhibits different biological activities through only small structural changes. Ideally, a biologically active molecule can be tracked and observed in a variety of biological and clinical settings in a non-invasive manner. As a step towards this goal, we report here a chemical synthesis of unnatural deep blue amino acid β-(1-azulenyl)-l alanine with different fluorescence and photophysical properties, which allows a spectral separation from the native tryptophan signal. This might be especially useful for cell localization studies and visualizing the targeted proteins. In particular, the synthesis of β-(1-azulenyl)-l alanine was achieved through a Negishi coupling which proved to be a powerful tool for the synthesis of unnatural tryptophan analogs. Upon β-(1-azulenyl)-l alanine incorporation into argyrin C, deep blue octapeptide variant was spectrally and structurally characterized.

Fluorescence Measurement of Kinetics of CheY Autophosphorylation with Small Molecule Phosphodonors.

The Escherichia coli chemotaxis protein CheY is a model receiver domain containing a native tryptophan residue that serves as a fluorescent probe for CheY autophosphorylation with small molecule phosphodonors. Here we describe fluorescence measurement of apparent bimolecular rate constants for reaction of wild type and mutant CheY with phosphodonors acetyl phosphate, phosphoramidate, or monophosphoimidazole. Step-by-step protocols to synthesize phosphoramidate (K+ salt) and monophosphoimidazole (Na+ salt), which are not commercially available, are provided. Key factors to consider in developing autophosphorylation assays for other response regulators are also discussed.

Using Tryptophan Mutants To Probe the Structural and Functional Status of BsSCO, a Copper Binding, Cytochrome c Oxidase Assembly Protein from Bacillus subtilis.

The synthesis of cytochrome c oxidase protein from Bacillus subtilis (i.e., BsSCO) binds copper with picomolar affinity, which increases the protein's melting temperature (i.e., TM) by 20 °C. Here two native tryptophans (i.e., W36 and W101) are identified as major contributors to BsSCO's structural form, and their contributions to the stability, intrinsic fluorescence, and copper binding properties of BsSCO are explored. Single mutations of tryptophan to phenylalanine decrease the TM by 10 °C and the folding free energy by 3-4 kcal/mol. A more severe change to alanine (i.e., W36A BsSCO) decreases the TM by 20 °C and the stability by 9 kcal/mol. However, these mutants bind copper with high affinity and assemble cytochrome c oxidase in vivo. Replacing phenylalanine at a position near (∼5 Å) the copper binding site with tryptophan (i.e., F42W) increases the TM of apo-BsSCO by 3 °C but diminishes the effect of copper binding. When both native tryptophans are changed to alanine, apo-BsSCO is unfolded in vitro and is not functional in cytochrome c oxidase assembly in vivo. A double-mutant of BsSCO in which W36A is combined with F42W exhibits a form of metastability. Apo-W36A/F42W BsSCO melts at 37 °C, which upon binding of copper shifts to 65 °C. B. subtilis expressing W36A/F42W BsSCO and grown at 37 °C does not assemble cytochrome c oxidase. However, when these cells are cooled to 25 °C, cytochrome c oxidase activity is recovered. Our results illustrate the subtle relationship between the structural stability and functional properties of BsSCO in the assembly of cytochrome c oxidase.

Search for DNA damage by human alkyladenine DNA glycosylase involves early intercalation by an aromatic residue

DNA repair enzymes recognize and remove damaged bases that are embedded in the duplex. To gain access, most enzymes use nucleotide flipping, whereby the target nucleotide is rotated 180° into the active site. In human alkyladenine DNA glycosylase (AAG), the enzyme that initiates base excision repair of alkylated bases, the flipped-out nucleotide is stabilized by intercalation of the side chain of tyrosine 162 that replaces the lesion nucleobase. Previous kinetic studies provided evidence for the formation of a transient complex that precedes the stable flipped-out complex, but it is not clear how this complex differs from nonspecific complexes. We used site-directed mutagenesis and transient-kinetic approaches to investigate the timing of Tyr162 intercalation for AAG. The tryptophan substitution (Y162W) appeared to be conservative, because the mutant protein retained a highly favorable equilibrium constant for flipping the 1,N6-ethenoadenine (ϵA) lesion, and the rate of N-glycosidic bond cleavage was identical to that of the wild-type enzyme. We assigned the tryptophan fluorescence signal from Y162W by removing two native tryptophan residues (W270A/W284A). Stopped-flow experiments then demonstrated that the change in tryptophan fluorescence of the Y162W mutant is extremely rapid upon binding to either damaged or undamaged DNA, much faster than the lesion-recognition and nucleotide flipping steps that were independently determined by monitoring the ϵA fluorescence. These observations suggest that intercalation by this aromatic residue is one of the earliest steps in the search for DNA damage and that this interaction is important for the progression of AAG from nonspecific searching to specific-recognition complexes.

Role of polysaccharide and lipid in lipopolysaccharide induced prion protein conversion

ABSTRACTConversion of native cellular prion protein (PrPc) from an α-helical structure to a toxic and infectious β-sheet structure (PrPSc) is a critical step in the development of prion disease. There are some indications that the formation of PrPSc is preceded by a β-sheet rich PrP (PrPβ) form which is non-infectious, but is an intermediate in the formation of infectious PrPSc. Furthermore the presence of lipid cofactors is thought to be critical in the formation of both intermediate-PrPβ and lethal, infectious PrPSc. We previously discovered that the endotoxin, lipopolysaccharide (LPS), interacts with recombinant PrPc and induces rapid conformational change to a β-sheet rich structure. This LPS induced PrPβ structure exhibits PrPSc-like features including proteinase K (PK) resistance and the capacity to form large oligomers and rod-like fibrils. LPS is a large, complex molecule with lipid, polysaccharide, 2-keto-3-deoxyoctonate (Kdo) and glucosamine components. To learn more about which LPS chemical constituents are critical for binding PrPc and inducing β-sheet conversion we systematically investigated which chemical components of LPS either bind or induce PrP conversion to PrPβ. We analyzed this PrP conversion using resolution enhanced native acidic gel electrophoresis (RENAGE), tryptophan fluorescence, circular dichroism, electron microscopy and PK resistance. Our results indicate that a minimal version of LPS (called detoxified and partially de-acylated LPS or dLPS) containing a portion of the polysaccharide and a portion of the lipid component is sufficient for PrP conversion. Lipid components, alone, and saccharide components, alone, are insufficient for conversion.

Reconstitution and Topological Analysis of Caveolin-1 in Bicelles

Caveolae are 50-100 nm invaginations in the plasma membrane of many cell types that play a critical role in signal transduction. Caveolin-1 is a 21 kD integral membrane protein that is required for the formation of caveolae. Caveolin-1 is thought to adopt an unusual horseshoe topology in the membrane where the N- and C- termini are cytoplasmic. Caveolin-1 has 4 native tryptophan residues in this region (W85, W98, W115, and W128) that can be used as reporters of the micro-environment of the polypeptide chain. Caveolin-1 single tryptophan mutants were successfully reconstituted into CHAPSO/DMPC bicelles. Fluorescence emission measurements were performed on each mutant and analysis of these spectra indicated that tryptophan residues 85 and 128 are in the head group region of the lipid bilayer with λmax values of 344.4 ± 2.4 nm and 338.2 ± 0.6 nm respectively, and tryptophan residues 98 and 115 are inserted into the hydrophobic core with λmax values of 334.4 ± 0.2 nm and 330.2 ± 1.0 nm, respectively. The information gleaned from these studies was supported by MD simulations performed on caveolin-1 in a DMPC bilayer. These data together support the postulation that caveolin-1 contains a membrane embedded turn.

The Membrane Catalysis Model: Apelin and its Receptor

Apelin is a peptide hormone that activates the class A G-protein coupled apelin receptor. Apelin is found in several bioactive isoforms in the body, ranging from 12 to 55 amino acids in length. Apelin-17 has previously been shown to bind to micelles of anionic detergents, which implies that apelin may interact with cell membranes prior to receptor activation. It has been theorized that this interaction with the membrane may induce conformational changes necessary for peptide recognition by the receptor, while increasing local concentration of the ligand in what is known as the membrane catalysis hypothesis. Here, we describe a method for conjugating various isoforms of apelin to fluorophores, while still maintaining bioactivity, as demonstrated by a phosphorylated extracellular signal-regulated kinase (pERK) assay in apelin receptor-transfected human embryonic kidney cells. These labeled peptides were then used for both fluorescence and diffusion-ordered nuclear magnetic resonance spectroscopy experiments in the presence of varying concentrations of micelle and bicelle species to test for membrane interaction as required by the membrane catalysis model. Forster resonance energy transfer experiments were performed with fluorescently labeled apelin isoforms to native tryptophan in fragments of the apelin receptor and to eGFP-tagged full-length apelin receptor to both localize the ligand-receptor binding interface and to quantify the peptide-receptor interaction.

Transthyretin Interacts with Amyloid-β Oligomers to Delay Amyloid Aggregation

Protective effects of Transthyretin (TTR) ameliorate Alzheimer's pathology in mice models. This suggests a possible route for cellular defense mechanism against toxic effects of protein misfolding and aggregation. This view is further supported by the results from in vitro experiments showing direct interactions between TTR and amyloid-β (Aβ) peptides. However, the molecular mechanism of the TTR-Aβ interactions is currently unknown primarily due to heterogeneities associated with self-association of both TTR and Aβ. Here we investigate the mechanism of TTR-Aβ interactions by examining the effects of WT-human, a monomeric mutant and murine TTR on the kinetics of aggregation of both Aβ1-40 and Aβ1-42. The three of forms of TTR differ in terms of stability as tetramers or monomers, e.g., the WT-human and murine TTR are primarily tetramers but the tetramers of murine TTR are more stable. To monitor the aggregation of Aβ but not of TTR we use a recently developed aggregation assay based on the fluorescence quenching of tetramethyl rhodamine (TMR)-labeled Aβ. Our data indicate that all three TTR variants delay aggregation of both Aβ1-40 and Aβ1-42. However, the effects are strongest for the monomeric mutant and weakest for the murine TTR. Kinetic measurements of fluorescence resonance energy transfer between native tryptophan residues of TTR and the EDANS-labeled Aβ indicate that binding of TTR is dependent on the aggregation status of Aβ with maximal binding occurring to larger aggregates of Aβ. Additionally, the modulatory effects of TTR are stronger on Aβ1-42 than on Aβ1-40 consistent with the known higher oligomerization propensity for Aβ1-42. Taken together our results indicate that the tetramers of WT-human TTR dissociates to monomers to bind to the aggregation intermediates of Aβ to subsequently slow down the growth of these intermediates.