Supramolecular approaches for protein framework modulation

Proteins are the building block of choice in nature to make complex, functional frameworks. The choice of 20 amino acids provides a rich diversity of sequence possibilities, allowing for the evolution of a wide range of structures that carry out unique functions. There is great interest in manufacturing highly ordered (supramolecular) protein architectures with a view to equal or exceed the structural properties demonstrated by nature’s examples. However, the structural diversity and complexity of proteins pose a challenge to controlled protein assembly. This thesis describes the use of macrocycles, macrocycle-metal complexes, mutagenesis, seeding, and crystallization to overcome this challenge, specifically exploiting supramolecular interactions between synthetic macrocyclic hosts and macromolecular guests. NMR spectroscopy and X-ray crystallography were used to observe host-guest interactions in solution and in the solid state. The model protein cytochrome c was cocrystallized with a complex of sulfonatothiacalix[4]arene (tsclx4) and zinc to yield two distinct crystal forms depending on the concentration of zinc. Further investigation was conducted with a histidine mutant of the trimeric protein RSL, yielding a dinuclear tsclx4/Zn complex, suggesting that the macrocycle-metal complex is an adaptable protein binder.1 Modulation of a previously described cubic assembly2 of sulfonato-calix[8]arene (sclx8) and RSL was carried out by surface charge modification and cross-seeding. Three mutants of RSL were generated to investigate the effect of replacing aspartic acid with asparagine. The goal was to alleviate charge repulsion with the acidic macrocycle and therefore enable cocrystallization at higher pH. Two cocrystal forms were obtained with one of the mutants, both requiring the same pH as the original structure. Control over which crystal form grew was enabled via seeding. The remaining mutants and wild type RSL were unaffected by cross-seeding in these conditions, suggesting replacement of the acidic side chain with an amide at this site is necessary for framework modulation. When this mutant was crystallized in the same conditions as native RSL,2 , an essentially identical assembly was observed. However, cocrystallization of a histidine mutant under these conditions resulted in an altered framework. The results reveal an attractive method for engineering protein frameworks.3 Complexation of sclx8 and Pent, a designed pentameric β-propeller lectin (developed by the Tawfik laboratory) was evaluated by using NMR spectroscopy and X-ray crystallography. Two crystal forms of the Pent – sclx8 complex were obtained, depending apparently on the ionic strength of the crystallization conditions. In conditions containing PEG as the sole precipitant, a 1:1 complex was obtained, where the calixarene forms multivalent interactions with the cationic protein surface. When the ionic strength is increased, a dimer of proteins sandwiches the calixarene. In this case the protein may be considered the host and the calixarene the guest. Strong binding to the concave, cationic pocket on Pent was observed in NMR, confirmed by a slow exchange process on the NMR time scale. This data,indicative of ~µM affinity, was consistent with the large protein – calixarene interface observed in the crystal structures. The calixarene scaffold, with its large surface area (~1500 Å2 for the pleated loop), is a versatile multivalent protein binder.4 The Pent – sclx8 system was further investigated with different crystallization conditions, yielding an icosahedral assembly of pentamers. This icosahedral assembly was confirmed using SAXS under similar conditions. The high porosity (∼70% solvent content) may have resulted in the lowresolution X-ray data of the crystals. However, Pent – sclx6 cocrystals provided evidence that sclx6 bound to the lysine-containing vertex of the pentamer. These data suggested that sclx8 may bind in a similar fashion, resulting in the formation of an icosahedron.5 Though this subject is still relatively new, much work has been done to understand protein – calixarene interactions. The results described in this thesis highlight the potential for macrocyclemediated protein assembly towards the fabrication of functional materials. Further work expanding on the concept of supramolecular synthons in protein – macrocycle frameworks may lead to precise framework fabrication across a range of different systems.

Funder

SSPC

Publisher

University of Galway

Rights

Attribution-NonCommercial-NoDerivatives 4.0 International

cbn

Collections

University of Galway Theses (PhD Theses)