The Rational Design of Environmentally-Responsive Elements in Synthetic Helical Fibers Open Access

Dublin, Steven Nathan (2007)

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Protein fibrils are among the principal synthetic targets for biomimetic materials design, primarily due to their frequent occurrence as major structural and functional components of native biological systems. The intricate morphological features and impressive array of functionalities exhibited by these native protein fibrils in biological systems has positioned these structures as vital models in the design of synthetic fibril systems which may be ultimately be utilized as functional components of nanoscale devices.

In this volume, a series of synthetic peptides are described which reversibly self assemble into well-defined fibrous materials in a manner similar to native protein fibrils.Furthermore, we have designed environmentally-responsive mechanisms into the peptide primary sequences, which couple the folding of the supramolecular structure to the ability to sense incremental changes in the local environmental conditions.We show that the assembly of the synthetic peptides into long aspect-ratio fibers occurs as a consequence of environmental conditions such as changes in pH, temperature, metal ion and small molecules concentration.

Table of Contents





Chapter I:Introduction

1.1 Motivation for developing biomimetic nanoscale materials

1.2 Self-assembly in native protein fibers

1.2.1 Minimalist structural motifs

1.2.2 β-strand inspired peptide assemblies

1.2.3 The α-helical structural subunit

1.3 Coiled-coil assemblies

1.3.1 a- and d-positions influence coiled-coil oligomerization state

1.3.2 e- and g-position residues

1.3.3 Solvent-accessible residues

1.3.4 Non-covalent interactions in native coiled-coil assembly

1.4 Established strategies for generating fibrous materials from

a-helical peptides

1.4.1 A hybrid strategy of magic number formulism and heptad elements

1.4.2 The rational design of a two-stranded, α-helical fiber

1.4.3 The generation of a triple-stranded, α-helical fiber

1.5 Summary

1.6 References

Chapter II: A pH responsive switch for helical fiber formation

2.1 Introduction

2.2 Results

2.2.1 Design of TZ1H peptide sequence

2.2.2 Circular dichroism (CD) spectrometry

2.2.3 Conformational changes in peptide TZ1H

2.2.4 Transmission Electron microscopy (TEM)

2.2.5 Cryo-electron microscopy tomography (CET)

2.2.6 High resolution scanning electron microscopy (HRSEM)

2.2.7 Modifications to TEM protocols

2.3 Summary

2.3.1 A depressed pKa value for the buried histidine side chain

2.3.2 Polar residues and thermostability of the TZ1H system

2.3.3 pH titration of e- and g-positions

2.4 Materials and methods

2.4.1 Peptide synthesis and purification

2.4.2 Circular dichroism

2.4.3 Electron microscopy

2.5 References

Chapter III: Extending the TZ1H peptide - A silver (I) ion-induced conformational switch

3.1 Introduction

3.1.1 Metal ion-binding sites are classified by pre-organization of ligands

3.1.2 Engineered metal-binding sites in polypeptide complexes

3.2 Results

3.2.1 Design of TZ1H peptide sequence

3.2.2 CD spectrometry of TZ1H in the presence of silver ion at pH 5.6

3.2.3 CD spectrometry of TZ1H peptide in the presence of sodium

thiosulfate and EDTA

3.2.4 Histidine residues are involved in the binding of silver (I) ion

3.2.5 Isothermal titration calorimetry (ITC)

3.2.6 Transmission electron microscopy (TEM) of the silver (I)

ion:TZ1H complex

3.2.7 Energy-dispersive X-ray (EDX) analysis provides evidence

for silver within the fiber

3.2.8 High angle annular dark field (HAADF) scanning transmission

electron microscopy (STEM) in Z-contrast imaging mode

3.2.9 Bright field low voltage STEM and backscatter secondary

electron microscopy

3.2.10Circular dichroism for TZ1H in the presence of zinc or copper

3.3 Summary

3.3.1 Metal ion-binding sites residing in internal cavities provide


3.4 Experimental section

3.4.1 Materials

3.4.2 Peptide synthesis and purification

3.4.3 Circular dichroism

3.4.4 Isothermal titration calorimetry

3.4.5 Transmission electron microscopy

3.4.6 Scanning transmission electron microscopy

3.4.7 Energy dispersive X-ray analysis

3.4.8 Amino acid analysis (AAA) and inductively coupled plasma massspectrometry (ICP-MS)

3.5 References

Chapter IV: Structural plasticity - Merging α-helix and β-sheet elements into a single peptide sequence

4.1 Introduction

4.2 Results

4.2.1 Design of TZ1T peptide sequence

4.2.2 Circular dichroism of TZ1T

4.2.3 Differential scanning calorimetry (DSC)

4.2.4 Transmission electron microscopy

4.2.5 Fourier transform infrared (FTIR) spectroscopy of TZ1T

4.2.6 Low-temperature scanning electron microscopy (LT-HRSEM)

4.3 Summary

4.4 Materials and methods

4.4.1 Peptide synthesis and purification

4.4.2 Circular dichroism

4.4.3 FTIR spectroscopy

4.4.4 Differential scanning calorimetry

4.4.5 Transmission electron microscopy

4.4.6 Low temperature high resolution scanning electron microscopy

4.5 References

Chapter V:Efforts toward engineering an allosteric switch into

an α-helical fiber

5.1 Introduction

5.2 Results

5.2.1 Design of first generation SKA peptide sequence

5.2.2 Circular dichroism of peptide SKA

5.2.3 Circular dichroism spectroscopy and TEM of peptide

S6KE solution

5.2.4 Circular dichroism of SKAE peptide

5.2.5 TEM of SKAE in the presence and absence of benzene

5.2.6 Small angle X-ray scattering measurements of

SKAE / S6KE fibers

5.2.7 Sedimentation equilibrium ultracentrifugation

5.3 Summary

5.3.1 Redesign of peptide S6K decreases fiber diameters

5.3.2 Partial disassembly and thermostability through ligand binding

5.3.3 Future improvements

5.4 Materials and methods

5.4.1 Peptide synthesis and purification

5.4.2 Small-angle X-ray scattering

5.4.3 Sedimentation equilibrium ultracentrifugation

5.5 References

Chapter VI:Conclusions and outlook

6.1 Fiber thickening

6.2 Future directions in discovering novel functionalities

6.3 References

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