Observing residual structure in disordered peptides with multidimensional infrared spectroscopy

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2012.Cataloged from PDF version of thesis.Includes bibliographical references.An estimated 35% of the human proteome is intrinsically disordered. Disordered proteins play a key role in physiologic and pathologic regulation, recognition, and signaling making protein disorder the subject of increasing investigation. Since disordered samples do not generate x-ray quality crystals and since they have conformations that interconvert faster than the time resolution of NMR or ESR, little is known about their structure or function. By combining isotope-edited two-dimensional infrared spectroscopy (2D IR) with spectral modeling based on molecular dynamics simulations, this work will show that one can measure the residual structure and conformational heterogeneity of a putatively disordered sequence. This methodology was first used to study the structure and dynamics of the tryptophan zipper 2 (TZ2) peptide. The TZ2 peptide is a 12 residue engineered sequence that employs the "tryptophan zipper" motif to stabilize a p-hairpin fold. For this work five isotopologues of TZ2 were synthesized, the p-turn label K8, the midstrand labels T10 and T3T1O, the N-terminal label S1, and the unlabeled peptide UL. Temperature dependent FTIR and 2D IR studies in conjunction with modeling revealed that the TZ2 peptide at low temperatures exists as a folded peptide with a type I' turn and a small previously unobserved subpopulation of a bulged loop structure. Upon heating a fraying of the peptides termini is observed, in addition the population of the type I' turn increases relative to the population of the bulged turn. The TZ2 peptide provided a model hairpin system to test the utility of different forms of analysis and isotope labeling patterns for the subsequent study of disordered hairpin peptides. This methodology was next employed to gain insight into the structure of the elastin-like peptide GVGVPGVG, a prototypical disordered system. For this work nine isotopologues were studied in addition to size dependent variants based on the VPGVG sequence and point mutants variants of the form GVGXPGVG. The conformational ensemble was found to contain a high population of irregular P-turns possessing two peptide hydrogen bonds to the proline C=O (see figure below). Further, it was found that this population grows as a function of 1) side-chain volume for the peptide series GVGXPGVG, where X = Gly, Ala, and Val, and 2) polymer size for the peptide series GVG(VPGVG)a, where n = 1-6 and 251. These findings provide new insight into the molecular origin of the mechanical properties of biopolymers containing XPG turns including collagen (X = Pro), elastin (X = Val), mussel byssus (X = Gly), dragline spider silk (X = Gly), and wheat glutenin (X = Gln).`by Joshua Lessing.Ph.D

STRETCHABLE CONDUCTIVE COMPOSITES FOR USE IN SOFT DEVICES

An elastically-deformable, conductive composite using elastomers and conductive fibers and simple fabrication procedures is provided. Conductive elastomeric composites offer low resistance to electrical current and are elastic over large (\u3e25%) extensional strains. They can be easily interfaced/built into structures fabricated from elastomeric polymers

Solvent and conformation dependence of amide I vibrations in peptides and proteins containing proline

We present a mixed quantum-classical model for studying the amide I vibrational dynamics (predominantly CO stretching) in peptides and proteins containing proline. There are existing models developed for determining frequencies of and couplings between the secondary amide units. However, these are not applicable to proline because this amino acid has a tertiary amide unit. Therefore, a new parametrization is required for infrared-spectroscopic studies of proteins that contain proline, such as collagen, the most abundant protein in humans and animals. Here, we construct the electrostatic and dihedral maps accounting for solvent and conformation effects on frequency and coupling for the proline unit. We examine the quality and the applicability of these maps by carrying out spectral simulations of a number of peptides with proline in D2O and compare with experimental observations.Netherlands Organization for Scientific Research (VIDI grant)National Science Foundation (U.S.) ((NSF) CHE-0911107

Melting of a beta-Hairpin Peptide Using Isotope-Edited 2D IR Spectroscopy and Simulations

Item does not contain fulltextIsotope-edited two-dimensional infrared spectroscopy has been used to characterize the conformational heterogeneity of the beta-hairpin peptide TrpZip2 (TZ2) across its thermal unfolding transition. Four isotopologues were synthesized to probe hydrogen bonding and solvent exposure of the beta-turn (K8), the N-terminus (S1), and the midstrand region (T10 and T3T10). Isotope-shifts, 2D lineshapes, and other spectral changes to the amide I 2D IR spectra of labeled TZ2 isotopologues were observed as a function of temperature. Data were interpreted on the basis of structure-based spectroscopic modeling of conformers obtained from extensive molecular dynamics simulations. The K8 spectra reveal two unique turn geometries, the type I' beta-turn observed in the NMR structure, and a less populated disordered or bulged loop. The data indicate that structures at low temperature resemble the folded NMR structure with typical cross-strand hydrogen bonds, although with a subpopulation of misformed turns. As the temperature is raised from 25 to 85 degrees C, the fraction of population with a type I' turn increases, but the termini also fray. Hydrogen bonding contacts in the midstrand region remain at all temperatures although with increasing thermal disorder. Our data show no evidence of an extended chain or random coil state for the TZ2 peptide at any temperature. The methods demonstrated here offer an approach to characterizing conformational variation within the folded or unfolded states of proteins and peptides

Elastomeric Tiles for the Fabrication of Inflatable Structures

This paper describes the fabrication of 3D soft, inflatable structures from thin, 2D tiles fabricated from elastomeric polymers. The tiles are connected using soft joints that increase the surface area available for gluing them together, and mechanically reinforce the structures to withstand the tensile forces associated with pneumatic actuation. The ability of the elastomeric polymer to withstand large deformations without failure makes it possible to explore and implement new joint designs, for example “double-taper dovetail joints,” that cannot be used with hard materials. This approach simplifies the fabrication of soft structures comprising materials with different physical properties (e.g., stiffness, electrical conductivity, optical transparency), and provides the methods required to “program” the response of these structures to mechanical (e.g., pneumatic pressurization) and other physical (e.g., electrical) stimuli. The flexibility and modularity of this approach is demonstrated in a set of soft structures that expanded or buckled into distinct, predictable shapes when inflated or deflated. These structures combine easily to form extended systems with motions dependent on the configurations of the selected components, and, when fabricated with electrically conductive tiles, electronic circuits with pneumatically active elements. This approach to the fabrication of hollow, 3D structures provides routes to new soft actuators.Chemistry and Chemical Biolog

Folding Analytical Devices for Electrochemical ELISA in Hydrophobic R H Paper

This work describes a device for electrochemical enzyme-linked immunosorbent assay (ELISA) designed for low-resource settings and diagnostics at the point of care. The device is fabricated entirely in hydrophobic paper, produced by silanization of paper with decyl trichlorosilane, and comprises two zones separated by a central crease: an embossed microwell, on the surface of which the antigen or antibody immobilization and recognition events occur, and a detection zone where the electrodes are printed. The two zones are brought in contact by folding the device along this central crease; the analytical signal is recorded from the folded configuration. Two proof-of-concept applications, an electrochemical direct ELISA for the detection of rabbit IgG as a model antigen in buffer and an electrochemical sandwich ELISA for the detection of malarial histidine-rich protein from Plasmodium falciparum (Pf HRP2) in spiked human serum, show the versatility of this device. The limit of detection of the electrochemical sandwich ELISA for the quantification of Pf HRP2 in spiked human serum was 4 ng mL–1 (102 pmol L–1), a value within the range of clinically relevant concentrations.Chemistry and Chemical Biolog

STRETCHABLE CONDUCTIVE COMPOSITES FOR USE IN SOFT DEVICES

An elastically-deformable, conductive composite using elastomers and conductive fibers and simple fabrication procedures is provided. Conductive elastomeric composites offer low resistance to electrical current and are elastic over large (\u3e25%) extensional strains. They can be easily interfaced/built into structures fabricated from elastomeric polymers