Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

Summary

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

Abstract

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities^1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils^3-8. A large variety of these peptides is commercially available.

This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a "beads on a string"-like structure by this process.⁹ The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

Introduction

Nature forms ordered and functional structures by the process of biomolecular self-assembly. Understanding the forces that govern this spontaneous process may lead to the ability to mimic self-assembly in vitro and consequently to major advances in the area of material sciences^10,11. Peptides, specifically, hold great promise as a biomolecular building block, since they present large structural diversity, ease of chemical synthesis, and can easily be functionalized with biological and chemical entities. The field of peptide self-assembly was pioneered by Ghadiri and his colleagues, who demonstrated the self-assembly of peptide nanotubes by cyclic p....

Protocol

1. Self-assembly of Homoaromatic Dipeptides

1. Weigh the desired peptide in its lyophilized form (e.g. NH₂-Phe-Phe-OH, Boc-Phe-Phe-COOH) and prepare a stock solution by dissolving the peptide in 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) to the appropriate concentration (e.g. 100 mg/ml for NH₂-Phe-Phe-OH and Boc-Phe-Phe-COOH)^7,17,46.
2. Mix the solution using vortex and place on the bench until the peptide is completely dissolved and the solut.......

Discussion

In summary, this paper demonstrates the ease in which peptide-based assemblies can be formed in vitro. The process involves commercially available peptides and solvents, and it occurs spontaneously under ambient conditions, upon the addition of a polar solvent to the test tube. It is crucial to use HFP as a solvent of the peptides, due to the low solubility of the peptides in other organic solvents. In addition, due to the high volatility of HFP it is necessary to prepare fresh stock solution for each experiment.......

Materials

Name	Company	Catalog Number	Comments
Name of Material/ Equipment	Company	Catalog Number	Comments/Description
NH2-Phe-Phe-OH	Bachem	G-2925.0001
Boc-Phe-Phe-OH	Bachem	A-3205.0005
1,1,1,3,3,3-hexafluoro-2-propanol	Sigma-Aldrich	52512-100ML
Ethanol absolute (Dehydrated) AR sterile	Bio-Lab Ltd.	52555	Blending with TDW for the preparation of 50% solution
Uranyl acetate	Sigma-Aldrich	73943	For negative staining. It is possible to work without it.
glass cover slip	Marienfeld Laboratory Glassware	110590
TEM grids	Electron Microscopy Sciences	FCF200-Cu-50	Formvar/Carbon 200 Mesh, Cu
Quantitive filter paper	Whatman	1001055
Deuterium Oxide (D2O)	Sigma-Aldrich	151882-100G	99.9 atom % D
CaF2 window	PIKE Technologies	160-1212	25 mm x 2 mm window. For FT-IR measurments
AFM tips	NanoScience Instruments	CFMR	Aspire probes, CFMR-25 series
Filter units	Millipore	SLGV033RS	Millex-GV, 0.22 μm, PVDF, 33 mm, gamma sterilized
SEM	FEI		Quanta 200 ESEM
TEM	FEI		Tecnai T12 G2 Spirit
AFM	JPK Instruments		A JPK NanoWizard3
FT-IR	Thermo Fisher Scientific		Nicolet 6700 advanced gold spectrometer
FT-IR Purge	Parker		BALSTON FT-IR Purge Gas Generator model 75-52
OMNIC (Nicolet) software	Thermo Nicolet Corporation		For FT-IR spectra analysis
Vortex mixer	Wisd Laboratory Equipment		ViseMix VM
Weight	Mettler Toledo		NewClassic MS
Sputter coater	Polaron		SC7640 Sputter Coater