Peptide In Buffer Research Articles

Steady-State and Time-Resolved Fluorescence Study of Selected Tryptophan-Containing Peptides in an AOT Reverse Micelle Environment.

The aim of this study was to demonstrate the utility of time-resolved fluorescence spectroscopy in the detection of subtle changes in the local microenvironment of a tryptophan chromophore in a confined and crowded medium of AOT reverse micelles, which mimic biological membranes and cell compartmentalization. For this purpose, fluorescence properties of L-tryptophan and several newly synthesized tryptophan-containing peptides in buffer and in an AOT reverse micelle medium were determined. It was shown that insertion of tryptophan and its short di- and tripeptides inside micelles led to evident changes in both the steady-state emission spectra and in fluorescence decay kinetics. The observed differences in spectral characteristics, such as a blue shift in the emission maxima, changes in the average fluorescence lifetime, and the appearance of environmental-dependent fluorescent species, showed the utility of time-resolved fluorescence spectroscopy as a sensitive tool for detecting subtle conformational modifications in tryptophan and its peptides induced by changes in polarity, viscosity, and specific interactions between chromophores and water molecules/polar groups/ions that occur inside reverse micelles.

Modular design of triple a-helical fibrillar peptides as an epitope delivery platform

Event Abstract Back to Event Modular design of triple α-helical fibrillar peptides as an epitope delivery platform Yaoying Wu1 and Joel H. Collier1 1 University of Chicago, Department of Surgery, United States Introduction: Self-assembly of peptides has been widely explored for biomaterials applications, including cell delivery, drug delivery, and vaccine platform. It was previously demonstrated that epitope-bearing β-sheet fibrillizing peptides could elicit strong and specific antibody response without supplemental immune adjuvants[1]. However, β-sheet fibrillizing peptides lack structural precision, and the kinetics of their assembly and disassembly are difficult to control. To address these shortcomings, we now report the design of a new self-assembly platform based on peptides that fold and assemble into α-helical nanofibers. Herein, we report our effort to develop a unique triple α-helical fibrillizing peptide system as vaccine delivery platform. Based upon Woolfson and coworkers’ previous works[2],[3], we engineered a self-assembling system composed of two peptides containing heterodimer and trimer domain (A+C and CB-), and one peptide as trimer (TriC). (Fig. 1) OVA epitope bearing peptide was synthesized via N-terminal extension on TriC peptide. This abstract reports the peptide design and self-assembly characteristics of this system. Materials and Methods: The peptides were synthesized using standard Fmoc solid-phase peptide synthesis, and purified via high performance liquid chromatography (HPLC), and marix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was utilized to confirm the molecular weight. Peptide fibers were formed by mixing equal molar of three peptides in 10 mM MOPS buffer at final concentration of 2 mM. The morphology of the self-assembled fiber was studied using both circular dichroism (CD) and transmission electron microscopy (TEM). Results and Discussion: A+C and CB- peptides are composed of both heterodimer and homotrimer sequences, while TriC peptide contains only the homotrimer sequence. Acidic and basic heterodimer peptides complement each other and function as overhanging “sticky-end” leading to fiber formation in our design, and the homotrimer sequence not only facilitates the fibrilization, but also provides a “docking position” for the epitope peptide on nanofibers. To incorporate epitopes in the fiber, OVA-TriC is synthesized by N-terminal extension with TriC as the anchor. The CB- and TriC did not self-assemble in the buffer, but the A+C peptide fibrilized in buffer possibly due to its β-sheet characteristics. (Fig. 2 A, B, C) Upon mixing the three peptides in buffer (ABC peptide assembly), nanofibers were formed after overnight incubation at room temperature. (Fig. 2D) The morphology of the obtained nanofibers was noticeably different from A+C peptide fiber, which was thicker and shorter (about 500 nm) according to TEM. Replacing TriC peptide by OVA-TriC peptide, the triple peptide system still self-assembled into nanofiber in buffer, despite of the presence of long epitope sequence. (Fig. 2 E, F) Moreover, the morphology of the obtained peptide fibers was not altered by the amount of OVA-TriC peptide in the mixture, indicating that the integration of OVA epitope did not significantly impact the fibrilization process. Fig. 1. Schematics for the design of triple α-helical fibrillar system. Left to right: helical-wheel diagram representing the heptad repeats of heterodimer sequence (acidic sequence, red; basic sequence blue) and homotrimer (green); schematic of the epitope-bearing fiber composed of three different peptides. Fig. 2. TEM images and CD plots of various peptides and mixtures: A) A+C peptide, B) CB- peptide, C) TriC peptide, D) ABC peptide assembly, E) ABC-1/2 OVA peptide assembly, F) ABC-OVA peptide assembly. Conclusion: Three α-helical peptides were designed to form nanofibers by overhanging “sticky-ends”. Neither CB- nor TriC self-assembled by itself in the MOPS buffer, only A+C peptide. However, mixing the three peptides in MOPS buffer led to formation of short nanofibers. It was also established that this triple peptide fibrilization system can tolerate OVA epitopes in a range of feeding ratios. This α-helical peptide system showed promising potential to modulate the peptide fiber length, and the dose of epitopes in nanofibers, both important in vaccine design. NIH/NIAID 5R01AI118182

A novel peptide nanomedicine for treatment of pancreatogenic diabetes

Pancreatogenic diabetes (PD) is a potentially fatal disease that occurs secondary to pancreatic disorders. The current anti-diabetic therapy for PD is fraught with adverse effects that can increase morbidity. Here we investigated the efficacy of novel peptide nanomedicine: pancreatic polypeptide (PP) in sterically stabilized micelles (SSM) for management of PD. PP exhibits significant anti-diabetic efficacy but its short plasma half-life curtails its therapeutic application. To prolong and improve activity of PP in vivo, we evaluated the delivery of PP in SSM. PP-SSM administered to rats with PD, significantly improved glucose tolerance, insulin sensitivity and hepatic glycogen content compared to peptide in buffer. The studies established the importance of micellar nanocarriers in protecting enzyme-labile peptides in vivo and delivering them to target site, thereby enhancing their therapeutic efficacy. In summary, this study demonstrated that PP-SSM is a promising novel anti-diabetic nanomedicine and therefore should be further developed for management of PD. From the Clinical EditorPancreatic peptide was earlier demonstrated to address pancreatogenic diabetes, but its short half-life represented major difficulties in further development for therapeutic use. PP-SSM (pancreatic polypeptide in sterically stabilized micelles) is a promising novel anti-diabetic nanomedicine that enables prolonged half-life and increased bioactivity of PP, as shown in this novel study, paving the way toward clinical studies in the near future.

An integrated on‐chip sirtuin assay

A microchip-based assay to monitor the conversion of peptide substrates by human recombinant sirtuin 1 (hSIRT1) is presented. For this purpose a fused silica microchip consisting of a microfluidic separation structure with an integrated serpentine micromixer has been used. As substrate for the assay, we used a 9-fluorenylmethoxycarbonyl (Fmoc)-labeled tetrapeptide derived from the amino acid sequence of p53, a known substrate of hSIRT1. The Fmoc group at the N-terminus resulting from solid-phase peptide synthesis enabled deep UV laser-induced fluorescence detection with excitation at 266 nm. The enzymatic reaction of 0.1 U/μL hSIRT1 was carried out within the serpentine micromixer using a 400 μM solution of the peptide in buffer. In order to reduce protein adsorption, the reaction channel was dynamically coated with hydroxypropylmethyl cellulose. The substrate and the deacetylated product were separated by microchip electrophoresis on the same chip. The approach was successfully utilized to screen various SIRT inhibitors.

Homocystamides promote free-radical and oxidative damage to proteins.

Elevated levels of homocysteine are associated with several major diseases. However, it is not clear whether homocysteine is a marker or a causative agent. The majority (ca. 80%) of the homocysteine present in humans is protein bound. The study of the posttranslational modification of proteins by homocysteine and its cyclic congener, homocysteine thiolactone, is emerging as an area of great current interest for unraveling the ongoing "mediator/marker controversy" [Jacobsen DW (2009) Clin Chem 55:1-2]. Interestingly, many of the pathologies associated with homocysteine are also linked to oxidative stress. In the current study, chemical evidence for a causal relationship between homocysteine-bound proteins and oxidative damage is presented. For example, a reproducible increase in protein carbonyl functionality occurs as a consequence of the reaction of human serum albumin with homocysteine thiolactone. This occurs at physiological temperature upon exposure to air without any added oxidants or free-radical initiators. Alpha-amino acid carbon-centered radicals, well-known precursors of protein carbonyls, are shown to form via a hydrogen atom transfer process involving thiolactone-derived homocystamides. Model peptides in buffer as well as native proteins in human blood plasma additionally exhibit properties in keeping with the homocystamide-facilitated hydrogen atom transfer and resultant carbon-centered radicals.

Proteomics Reveals a Physical and Functional Link between Hepatocyte Nuclear Factor 4α and Transcription Factor IID

Proteomic analyses have contributed substantially to our understanding of diverse cellular processes. Improvements in the sensitivity of mass spectrometry approaches are enabling more in-depth analyses of protein-protein networks and, in some cases, are providing surprising new insights into well established, longstanding problems. Here, we describe such a proteomic analysis that exploits MudPIT mass spectrometry and has led to the discovery of a physical and functional link between the orphan nuclear receptor hepatocyte nuclear factor 4alpha (HNF4alpha) and transcription factor IID (TFIID). A systematic characterization of the HNF4alpha-TFIID link revealed that the HNF4alpha DNA-binding domain binds directly to the TATA box-binding protein (TBP) and, through this interaction, can target TBP or TFIID to promoters containing HNF4alpha-binding sites in vitro. Supporting the functional significance of this interaction, an HNF4alpha mutation that blocks binding of TBP to HNF4alpha interferes with HNF4alpha transactivation activity in cells. These findings identify an unexpected role for the HNF4alpha DNA-binding domain in mediating key regulatory interactions and provide new insights into the roles of HNF4alpha and TFIID in RNA polymerase II transcription.

Amyloid‐forming peptides from fungal adhesins

Candida albicans Als adhesins bind peptide ligands and mediate cell aggregation with amyloid properties. A 13‐residue peptide, Als5p322‐334, has a 90% β‐aggregation potential and forms amyloids, as do truncated versions of the Als5 protein (Otoo et al, Euk Cell 7: 776‐782, 2008). We therefore searched in other fungal adhesins for β‐aggregation forming sequences with the TANGO algorithm. Two additional adhesion peptides, from C. albicans Eap1p117‐133 and Saccharomyces cerevisiae Flo1p305‐315 have aggregation potentials of 90% and 42% respectively. Peptide interactions were analyzed by circular dichroism, absorbance and fluorescence spectroscopy to monitor secondary structure and amyloid formation. CD spectra showed random coil for both Eap1 and Flo1 peptides in buffer, changing to β‐aggregate with an ellipticity minimum at 230‐235nm after stirring to induce amyloid formation. The stirred solutions of both Flo1 and Eap1 peptides showed an increase in Congo Red absorbance with a shoulder near 550nm, and also had greatly enhanced thioflavin‐T fluorescence. These results with the Flo1 and Eap1 peptides suggest a change from non‐amyloid characteristics when initially dissolved in buffer to amyloid formation with stirring. Therefore, sequences from two additional adhesins show conformational changes leading to amyloid formation. Supported by SC1 GM083756.

Amyloid Formation By Peptides From Yeast Adhesins

Candida albicans adhesins bind to peptides and lead to cell aggregation with amyloid properties. We therefore searched in other fungal adhesins for β-aggregation forming sequences with the TANGO algorithm and synthesized three peptides. The C. albicans Als5 peptide, SNG (SNGIVIVATTRTV), has a 90% β-aggregation potential and forms amyloids (Otoo, et al, Euk. Cell, 7(5): 776-782, 2008). The two additional peptides, from C. albicans Eap1 adhesin (HTAVTTGVTIITVTTND) and Saccharomyces cerevisiae Flo1 adhesin (TDETVIVIRTP), have aggregation potentials of 90% and 42% respectively, and were studied for amyloid formation.Peptide interactions were analyzed by circular dichroism (CD), absorbance and fluorescence spectroscopy to monitor secondary structure and amyloid formation. CD spectra showed unstructured random coil for both Eap1 and Flo1 peptides in buffer, changing to β-aggregate with an ellipticity minimum at 230-235nm after stirring to induce amyloid formation. The stirred solutions of both Flo1 and Eap1 peptides showed an increase in Congo Red absorbance with a shoulder near 550nm and also had enhanced thioflavin-T fluorescence. Thioflavin-T emission intensity is much greater when it is bound to amyloids and the emission spectra of both amyloid peptides show a significant fluorescence intensity increase. These results with the Flo1 and Eap1 peptides suggest a change from non-amyloid characteristics when initially dissolved in buffer to amyloid formation with stirring. Therefore, sequences from two additional adhesins show conformational changes leading to amyloid formation.

Enzymatic Properties of Purified Murine Fatty Acid Transport Protein 4 and Analysis of Acyl-CoA Synthetase Activities in Tissues from FATP4 Null Mice

Fatty acid transport protein 4 (FATP4) is an integral membrane protein expressed in the plasma and internal membranes of the small intestine and adipocyte as well as in the brain, kidney, liver, skin, and heart. FATP4 has been hypothesized to be bifunctional, exhibiting both fatty acid transport and acyl-CoA synthetase activities that work in concert to mediate fatty acid influx across biological membranes. To determine whether FATP4 is an acyl-CoA synthetase, the murine protein was engineered to contain a C-terminal FLAG epitope tag, expressed in COS1 cells via adenovirus-mediated infection and purified to near homogeneity using alpha-FLAG affinity chromatography. Kinetic analysis of the enzyme was carried out for long chain (palmitic acid, C16:0) and very long chain (lignoceric acid, C24:0) fatty acids as well as for ATP and CoA. FATP4 exhibited substrate specificity for C16:0 and C24:0 fatty acids with a V(max)/K(m) (C16:0)/V(max)/K(m) (C24:0) of 1.5. Like purified FATP1, FATP4 was insensitive to inhibition by triacsin C but was sensitive to feedback inhibition by acyl-CoA. Although purified FATP4 exhibited high levels of palmitoyl-CoA and lignoceroyl-CoA synthetase activity, extracts from the skin and intestine of FATP4 null mice exhibited reduced esterification for C24:0, but not C16:0 or C18:1, suggesting that in vivo, defects in very long chain fatty acid uptake may underlie the skin disorder phenotype of null mice.

An apolipoprotein AI mimetic peptide: membrane interactions and the role of cholesterol.

The 18-amino acid amphipathic helical peptide Ac-DWFKAFYDKVAEKFKEAF-NH(2) promotes the separation of cholesterol from the phospholipid, resulting in the formation of cholesterol crystallites, even at mole fractions of cholesterol as low as 0.3. The peptide exerts a greater degree of penetration into membranes of pure phosphatidylcholine in the absence of cholesterol than into bilayers of phosphatidylcholine and cholesterol. The circular dichroism spectrum of the peptide in buffer indicates that it self-associates, leading to the formation of structures with higher helical content. However, in the presence of lipid, the peptide remains helical over a larger concentration range. The peptide undergoes a thermal transition on heating. Cholesterol has little effect on the secondary structure of the peptide; however, increased Trp emission intensity in the absence of cholesterol indicates a deeper penetration of the helix upon removal of cholesterol from the membrane. The results with these model systems demonstrate changes in peptide-lipid interactions that may be related to the observed biological properties of this peptide.

Differential expression of endophilin 1 and 2 dimers at central nervous system synapses.

Endophilin 1 is proposed to participate in synaptic vesicle biogenesis through SH3 domain-mediated interactions with the polyphosphoinositide phosphatase synaptojanin and the GTPase dynamin. Endophilin family members have also been identified as binding partners for a number of diverse cellular proteins. We define here the endophilin 1-binding site within synaptojanin 1 and show that this sequence independently and selectively purifies from brain extracts endophilin 1 and a closely related protein, endophilin 2. Endophilin 2, like endophilin 1, is highly expressed in brain, concentrated in nerve terminals, and found in complexes with synaptojanin and dynamin. Although a fraction of endophilins 1 and 2 coexist in the same complex, the distribution of these endophilin isoforms among central synapses only partially overlaps. Endophilins 1 and 2 are found predominantly as stable dimers through a predicted coiled-coil domain in their conserved NH2-terminal moiety. Dimerization may allow endophilins to link a number of different cellular targets to the endocytic machinery.

Sites in the A2 Subunit Involved in the Interfactor VIIIa Interaction

Sites in the A2 Subunit Involved in the Interfactor VIIIa Interaction

Solution conformation of a peptide corresponding to bovine kappa-casein B residues 130-153 by circular dichroism spectroscopy and 1H-nuclear magnetic resonance spectroscopy.

The peptide Pro130-Thr-Ser-Thr-Pro-Thr-Ile-Glu-Ala-Val-Glu140- Ser-Thr-Val-Ala-Thr-Leu-GLu-Ala-Ser-Pro150-Glu-Val-Ile, which corresponds to residues 130-150 of kappa-casein B, was synthesized and the conformation of the peptide in solution investigated by circular dichroism (CD) spectroscopy, structure prediction algorithms and 1H-nuclear magnetic resonance spectroscopy. In a solution containing the structure-enhancing solvent trifluoroethanol the CD spectrum was typical of a peptide in the alpha-helical conformation and nuclear magnetic resonance showed that the amino acids between Ile136 and Ser149 (kappa-casein numbering) were predominantly in the alpha-helical conformation but that Pro130 to Thr135 and Pro150 to Ile153 were not. In addition, Thr133-Pro134 and Ser-149-Pro150 were primarily in the trans conformation, the residues from Thr131 to Thr135 were in unordered structures and the residues from Glu151 to Ile153 were in an extended conformation. Residues Glu137 to Glu140 and Thr145 to Ala148 also displayed some 3(10)-helix character. When the peptide was dissolved in 10 mM-cetyltrimethylammonium chloride solution at pH 6, the CD spectra indicated that the proportion of helical structure was comparable to that of the peptide in trifluoroethanol solution (400 ml/l), whereas when the peptide was dissolved in buffer alone in 10 mM-SDS solution, the CD spectra were consistent with a low helical content. Acidification of these solutions to pH 2.85 resulted in a slight increase in the helical content of the peptide in buffer and more markedly in buffer containing SDS. When the peptide was in 5 mM-CaCl2 solution at neutral pH, the CD spectrum indicated that some ordered structure was present. Taken together these results indicate that the ionizable residues Glu137, Glu140, Glu147 and Glu151 could be important in determining the stability of the putative helix. The structure predictions found that the sequence from Glu137 to Pro150 would be more likely to be in a helical than any other conformation in the intact bovine protein, but that pig, sheep and goat kappa-caseins did not give a prediction of a strongly helical region in this part of the molecule.

Membrane-binding amphipathic alpha-helical peptide derived from CTP:phosphocholine cytidylyltransferase.

A peptide corresponding to a portion of the amphipathic alpha-helical region of CTP:phosphocholine cytidylyltransferase was synthesized. This region of the enzyme was proposed to be the membrane-binding domain [Kalmar, G.B., Kay, R.J., Lachance, A., Aebersold, R., & Cornell, R.B. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6029]. We have shown that the peptide is physically associated with PG vesicles. CD of the peptide in buffer suggested a primarily random structure, while, in the presence of trifluoroethanol, the peptide was alpha-helical. Anionic lipid vesicles promoted an alpha-helical conformation, whereas neutral or cationic lipid vesicles did not alter the random structure of the peptide, suggesting a selective stabilization of the alpha-helix by anionic membranes. The fluorescence of the single tryptophan residue, which lies on the hydrophobic face of the amphipathic alpha-helix, was studied. Anionic lipid vesicles specifically induced a shift in the fluorescence to a lower wavelength. Fluorescence quenching by the aqueous-phase quencher, I-, and the lipid-phase quencher 9,10-dibromo-PC was used to determine the accessibility of the tryptophan to each of these environments. The presence of anionic lipid vesicles, but not nonanionic lipid vesicles, decreased the quenching by I- suggesting that, in the presence of anionic lipids, the tryptophan residue is poorly accessible to the aqueous I-. Dibromo-PC significantly quenched the fluorescence only when present in anionic vesicles, confirming the membrane location of the tryptophan residue and the lipid specificity of this interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth-inhibitory and bactericidal effects of human parotid salivary histidine-rich polypeptides on Streptococcus mutans.

Growth inhibition and cell viability assays demonstrate that the histidine-rich polypeptides isolated from human parotid saliva are bacteriostatic and bactericidal for strains of Streptococcus mutans belonging to the serotype b and c classifications. Both inhibition of growth and cell division are enhanced by preincubation of bacteria with these polypeptides in low-ionic-strength buffers of acidic and neutral pH before dilution into enriched growth media. With prior exposure at pH 6.8, inhibition by these polypeptides of the serotype c strains, S. mutans GS5 and SB, as well as the serotype b strain, S. mutans BHT, is reversible over time under the experimental conditions selected. With similar exposure at pH 5.2, however, irreversible damage is manifested by complete inhibition of both growth and cell viability. At concentrations of 250 micrograms of the mixture of histidine-rich polypeptides per 5 X 10(5) bacterial cells per ml in the acidic preincubation buffer, bacterial lethality is maintained for a period of 48 h in the enriched growth media. At a 50-micrograms/ml concentration of these salivary agents, approximately 80% killing of S. mutans SB is noted after a 24-h incubation; however, surviving bacteria multiply and reach turbidities of untreated control cells when examined at the 48-h growth point. Similarly, hen egg white lysozyme is also found to be bactericidal for these microorganisms when preincubation is carried out under acidic conditions. However, in contrast to the histidine-rich polypeptides, lysozyme under these experimental conditions does not inhibit growth of S. mutans SB at neutral pH, although it does inhibit growth of both S. mutans BHT and S. mutans GS5 at this pH. Preexposure of S. mutans SB to the peptides in buffer at ionic strengths of 0.025 to 0.125, followed by either viability assays under nongrowing conditions or growth inhibition studies, suggests that there is very little effect of ionic strength on the antibacterial function of these peptides. In contrast to the inhibition of viability noted under growing conditions, lower concentrations of the histidine-rich polypeptides were required to elicit immediate cell death under nongrowing conditions.