Putting a new spin on spider silk.
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Alanine↗
Biomedical subjects
D A Tirrell
Publications and source records attributed to D A Tirrell.
Chemical sequence control of beta-sheet assembly in macromolecular crystals of periodic polypeptides.
A family of uniform periodic polypeptides has been prepared by bacterial expression of the corresponding artificial genes, with the objective of exploring the potential for control of supramolecular organization in genetically engineered protein-based polymeric materials. The repeating units of the polypeptides consist of oligomeric alanyl-glycine sequences interspersed with glutamic acid residues inserted at intervals of 8 to 14 amino acids. Crystallization of such materials from formic acid produces beta-sheet structures in the solid state, as shown by vibrational spectroscopy, nuclear magnetic resonance spectroscopy, and wide-angle x-ray diffraction. The diffraction results, together with observations from electron microscopy, are consistent with the formation of needle-shaped lamellar crystals whose thickness is controlled by the periodicity of the primary sequence. These results can be used to control solid-state structure in macromolecular materials.
Amino Acid Sequence↗
Membrane solubilization by a hydrophobic polyelectrolyte: surface activity and membrane binding.
We have previously observed that the hydrophobic polyelectrolyte poly(2-ethylacrylic acid) solubilizes lipid membranes in a pH-dependent manner, and we have exploited this phenomenon to prepare lipid vesicles that release their contents in response to pH, light, or glucose (Thomas, J. L., and D. A. Tirrell. Acc. Chem. Res. 25:336-342, 1992). The physical basis for the interaction between poly(2-ethylacrylic acid) and lipid membranes has been explored using surface tensiometry and fluorimetry. Varying the polymer concentration results in changes in surface activity and membrane binding that correlate with shifts in the critical pH for membrane solubilization. Furthermore, the binding affinity is reduced as the amount of bound polymer increases. These results are consistent with a hydrophobically driven micellization process, similar to those observed with apolipoproteins, melittin, and other amphiphilic alpha-helix-based polypeptides. The absence of specific secondary structure in the synthetic polymer suggests that amphiphilicity, rather than structure, is the most important factor in membrane micellization by macromolecules.
Acrylates↗
Signal-sensitive release of content from liposomes bearing surface-grafted polyelectrolyte chains.
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Electrolytes↗
Photoregulation of the binding of an azobenzene-modified poly(methacrylic acid) to phosphatidylcholine bilayer membranes.
Radical copolymerization of a 4:1 mixture of methacrylic acid and N-[4-(phenylazo)-phenyl]methacrylamide afforded a copolymer of methacrylic acid bearing 10.4 mol% pendent azobenzene units. The copolymer was sensitive to irradiation by virtue of the photochemical trans-to-cis isomerization of the pendent chromophore. The pH-dependent conformational transition characteristic of poly(methacrylic acid) was shifted to a higher pH by the hydrophobic nature of the azobenzene units, but viscosity measurements revealed no further shift in the transition upon photoisomerization. The potentiometric titration curves of the two photoisomeric states of the chain were displaced in a manner consistent with the enhanced polarity of the cis form of the chromophore. The adsorption of the chain on phosphatidylcholine bilayer membranes was strikingly sensitive to light; at pH 7, the cis form adsorbed weakly if at all, whereas the trans form bound strongly to the membrane surface with concomitant reorganization of the lipid bilayer structure.
Azo Compounds↗
Tris buffer causes acyl chain interdigitation in phosphatidylglycerol.
The structure of the gel phase and the properties of the acyl chain disordering transition of dipalmitoyl phosphatidylglycerol (DPPG) have been studied using differential scanning calorimetry, differential scanning dilatometry, and X-ray diffraction. In the presence of small, monovalent cations, DPPG at 22 degrees C exists in a lamellar phase in which the hydrocarbon chains are tilted from the perpendicular to the bilayer surface. Around 34 degrees C, there is a small pretransition (delta H less than 1 kcal/mol) followed by the main transition at 40.4 degrees C (delta H = 8.3 kcal/mol; delta V = 0.0381 ml/g). If DPPG is suspended in Tris-HCl buffer in the absence of other monovalent cations, X-ray diffraction data show that at 22 degrees C, the gel phase consists of interdigitated acyl chains perpendicular to the plane of the bilayer. No pretransition is observed and the main transition occurs at 41.3 degrees C with delta H = 9.1 kcal/mol and delta V = 0.0514 ml/g. If sufficient Na+ or K+ ions are added to the Tris-buffered DPPG, the phase behavior reverts to what is observed in the absence of Tris. Analysis of the energetics of the main transition shows that the increase in van der Waals interaction energy resulting from the larger delta V in Tris can be compensated by the favorable energetics of removing terminal methyl groups from the bilayer surface. The amount of disordering, i.e. formation of gauche rotamers, is likely to be the same in Tris as it is in buffers without amphiphilic cations.
Calorimetry, Differential Scanning↗
pH sensitization of phospholipid vesicles via complexation with synthetic poly(carboxylic acid)s.
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Carboxylic Acids↗
Peptide-derived self-assembled monolayers: adsorption of N-stearoyl L-cysteine methyl ester on gold.
The work reported herein concerns the assembly of N-stearoyl L-cysteine methyl ester [CH3(CH2)16COCysOMe, 1] on the surface of gold. This compound serves as a simple model of a related polypeptide, which has been designed to adopt a beta-sheet architecture on metallic and oxide surfaces. We describe the preparation of monolayers of 1, and characterization of these layers via ellipsometry, vibrational spectroscopy and X-ray photoelectron spectroscopy. The results are most consistent with a disordered array of the alkyl chains, in which close packing is frustrated by a mismatch in the cross-sectional areas of the cysteinyl ester head group and the stearoyl chains of the thiol. Despite the disorder, the alkyl chains form a hydrophobic surface layer, with an advancing contact angle for water comparable to that observed for octadecanethiol on gold.
Adsorption↗
Hemolytic activity of pH-responsive polymer-streptavidin bioconjugates.
Drug delivery systems that increase the rate and/or quantity of drug release to the cytoplasm are needed to enhance cytosolic delivery and to circumvent nonproductive cell trafficking routes. We have previously demonstrated that poly(2-ethylacrylic acid) (PEAAc) has pH-dependent hemolytic properties, and more recently, we have found that poly(2-propylacrylic acid) (PPAAc) displays even greater pH-responsive hemolytic activity than PEAAc at the acidic pHs of the early endosome. Thus, these polymers could potentially serve as endosomal releasing agents in immunotoxin therapies. In this paper, we have investigated whether the pH-dependent membrane disruptive activity of PPAAc is retained after binding to a protein. We did this by measuring the hemolytic activity of PPAAc-streptavidin model complexes with different protein to polymer stoichiometries. Biotin was conjugated to amine-terminated PPAAc, which was subsequently bound to streptavidin by biotin complexation. The ability of these samples to disrupt red blood cell membranes was investigated for a range of polymer concentrations, a range of pH values, and two polymer-to-streptavidin ratios of 3:1 and 1:1. The results demonstrate that (a) the PPAAc-streptavidin complex retains the ability to lyse the RBC lipid bilayers at low pHs, such as those existing in endosomes, and (b) the hemolytic ability of the PPAAc-streptavidin complex is similar to that of the free PPAAc.
Acrylates↗
Chemical and biosynthetic approaches to the production of novel polypeptide materials.
Three approaches to the synthesis of the repetitive copolypeptide [(GlyAla)3-GlyProGlu]n (1) are described. Direct chemical synthesis of 1 via classical solution methods required 18 steps and afforded a polydisperse product with an average molecular weight of less than 10,000. Two alternative genetic strategies were also explored. In the first, chemically synthesized DNA oligomers were self-ligated to produce a population of multimers, which were fitted with translational start and stop signals and inserted into an expression plasmid containing the lambda PL promoter and a synthetic ribosome binding site. Transformation of E. coli led to the isolation of a stable recombinant plasmid carrying an insert encoding 12 repeats of sequence 1. Attempts to identify polypeptide 1 after induction of transformed cultures were unsuccessful. A second strategy, generating a tripartite derivative of sequence 1 carrying short N- and C-terminal extensions, afforded excellent yields of product. The relative merits of chemical and genetic approaches to repetitive polypeptide materials are discussed.
Amino Acid Sequence↗