http://ctrstbio.org.uic.edu/manuals/kellybba.pdf

How to study proteins by circular dichroism

This tutorial review looks at the design rules that allow peptides to be exploited as building blocks for the assembly of nanomaterials. These design rules are either derived by copying nature (α-helix, β-sheet) or may exploit entirely new designs based on peptide derivatives (peptide amphiphiles, π-stacking systems). We will examine the features that can be introduced to allow self-assembly to be controlled and directed by application of an externally applied stimulus, such as pH, light or enzyme action. Lastly the applications of designed self-assembly peptide systems in biotechnology (3D cell culture, biosensing) and technology (nanoelectronics, templating) will be examined.

Designing peptide based nanomaterials

Challenges and breakthroughs in recent research on self-assembly

Kepa Ruiz-Mirazo,†,∥ Carlos Briones,‡,∥ and Andreś de la Escosura* †Biophysics Unit (CSIC-UPV/EHU), Leioa, and Department of Logic and Philosophy of Science, University of the Basque Country, Avenida de Tolosa 70, 20080 Donostia−San Sebastiań, Spain ‡Department of Molecular Evolution, Centro de Astrobiología (CSIC−INTA, associated to the NASA Astrobiology Institute), Carretera de Ajalvir, Km 4, 28850 Torrejoń de Ardoz, Madrid, Spain Organic Chemistry Department, Universidad Autońoma de Madrid, Cantoblanco, 28049 Madrid, Spain

Prebiotic systems chemistry: new perspectives for the origins of life.

Peptide-based hydrogels are an important class of biomaterials finding use in food industry and potential use in tissue engineering, drug delivery and microfluidics. A primary experimental method to explore the physical properties of these hydrogels is rheology. A fundamental understanding of peptide hydrogel mechanical properties and underlying molecular mechanisms is crucial for determining whether these biomaterials are potentially suitable for biotechnological uses. In this critical review, we cover the literature containing rheological characterization of the physical properties of peptide and polypeptide-based hydrogels including hydrogel bulk mechanical properties, gelation mechanisms, and the behavior of hydrogels during and after flow (219 references).

Rheological properties of peptide-based hydrogels for biomedical and other applications.

Coiled-coil motifs provide simple systems for studying molecular self-assembly. We designed two 28-residue peptides to assemble into an extended coiled-coil fiber. Complementary interactions in the core and flanking ion-pairs were used to direct staggered heterodimers. These had sticky-ends to promote the formation of long fibers. For comparison, we also synthesized a permuted version of one peptide to associate with the other peptide and form canonical heterodimers with blunt-ends that could not associate longitudinally. The assembly of both pairs was monitored in solution using circular dichroism spectroscopy. In each case, mixing the peptides led to increased and concentration-dependent circular dichroism signals at 222 nm, consistent with the desired -helical structures. For the designed fiber-producing peptide mixture, we also observed a linear dichroism effect during flow orientation, indicative of the presence of long fibrous structures. X-ray fiber diffraction of partially aligned samples gave patterns indicative of coiled-coil structure. Furthermore, we used electron microscopy to visualize fiber formation directly. Interestingly, the fibers observed were at least several hundred micrometers long and 20 times thicker than expected for the dimeric coiled-coil design. This additional thickness implied lateral association of the designed structures. We propose that complementary features present in repeating structures of the type we describe promote lateral assembly, and that a similar mechanism may underlie fibrillogenesis in certain natural systems.

http://www.chm.bris.ac.uk/org/woolfson/papers/paper19.pdf

Sticky-end assembly of a designed peptide fiber provides insight into protein fibrillogenesis.

Two stages in the rational redesign of a peptide-based, self-assembling fiber (SAF) are described. The SAF system comprises two peptides designed to form an offset a-helical coiled-coil heterodimer. The "sticky-ends" are complementary and promote longitudinal assembly. Alone, the two peptides are unstructured, but co-assemble upon mixing to form a-helical fibrils, which bundle to form fibers 40-50 nm wide and tens of micrometers long. Assembly is controllable and occurs at pH7 in water, mak- ing SAFs a potential scaffold for 3D cell culture. The purposes of the redesigns were 1) to investigate the fiber-thickening pro- cess, and 2) to increase fiber stability for potential biological and biomedical applications. First, mutations were made to the original peptide designs to increase fibril-fibril interactions and so produce thicker and more-stable fibers. The second iteration aimed to increase the primary peptide-peptide interactions by increasing the overlap in the offset dimer and so promote the initial step in fiber formation. As judged by circular dichroism spectroscopy and transmission electron microscopy, both itera- tions improved fiber assembly and stability: the critical peptide concentration for assembly improved from 60 lM to 4 lM; the midpoint of thermal unfolding increased from 22°C to 65°C; and the salt tolerance improved from 75 mM to greater than 250 mM KCl. These improvements bring closer applications of the SAF system under physiological conditions, for example as a biocompatible material for 3D cell culture. In addition, ordered surface features were observed in the second- and third-genera- tion fibers compared with the original design. This indicates improved internal order in the redesigned fibers. In turn, this sug- gests a molecular mechanism for the improved stability and sheds light on the fiber-assembly process.

http://www.chm.bris.ac.uk/org/woolfson/papers/paper48.pdf

Engineering Increased Stability into Self-Assembled Protein Fibers

To improve our understanding of conformational transitions in proteins, we are attempting the de novo design of peptides that switch structural state. Here, we describe coiled-coil peptides with sequence and structural duality; that is, features compatible with two different coiled-coil motifs superimposed within the same sequence. Specifically, we promoted a parallel leucine-zipper dimer under reducing conditions, and a monomeric helical hairpin in an intramolecularly disulfide bridged state. Using an iterative process, we engineered peptides that formed stable structures consistent with both targets under the different conditions. Finally, for one of the designs, we demonstrated a one-way switch from the helical hairpin to the coiled-coil dimer upon addition of disulfide-reducing agents.

http://www.chm.bris.ac.uk/org/woolfson/papers/paper41.pdf

Sequence and Structural Duality: Designing Peptides to Adopt Two Stable Conformations

Coiled-coil motifs provide simple systems for studying molecular self-assembly. We designed two 28-residue peptides to assemble into an extended coiled-coil fiber. Complementary interactions in the core and flanking ion-pairs were used to direct staggered heterodimers. These had \"sticky-ends\" to promote the formation of long fibers. For comparison, we also synthesized a permuted version of one peptide to associate with the other peptide and form canonical heterodimers with \"blunt-ends\" that could not associate longitudinally. The assembly of both pairs was monitored in solution using circular dichroism spectroscopy. In each case, mixing the peptides led to increased and concentration-dependent circular dichroism signals at 222 nm, consistent with the desired alpha-helical structures. For the designed fiber-producing peptide mixture, we also observed a linear dichroism effect during flow orientation, indicative of the presence of long fibrous structures. X-ray fiber diffraction of partially aligned samples gave patterns indicative of coiled-coil structure. Furthermore, we used electron microscopy to visualize fiber formation directly. Interestingly, the fibers observed were at least several hundred micrometers long and 20 times thicker than expected for the dimeric coiled-coil design. This additional thickness implied lateral association of the designed structures. We propose that complementary features present in repeating structures of the type we describe promote lateral assembly, and that a similar mechanism may underlie fibrillogenesis in certain natural systems.

Sticky-end assembly of a designed peptide fibre provides insight into protein fibrillogenesis

This invention relates to protein fibre formation and in particular to methods of producing protein fibres to form a protein structure comprising a plurality of first polypeptide units arranged in a first polypeptide strand and a plurality of second polypeptide units arranged in a second polypeptide strand, the strands preferably forming a coiled coil structure, and in which a first polypeptide unit in the first strand extends beyond a corresponding second polypeptide unit in the second strand in the direction of the strands.

Maya J. Pandya

Papers

Sticky-end assembly of a designed peptide fiber provides insight into protein fibrillogenesis.

Engineering Increased Stability into Self-Assembled Protein Fibers

Sequence and Structural Duality: Designing Peptides to Adopt Two Stable Conformations

Sticky-end assembly of a designed peptide fibre provides insight into protein fibrillogenesis

Protein structures and protein fibres