Engineering synthetic Lsm rings for applications in nanotechnology
thesisposted on 2022-03-28, 16:36 authored by Francesca Manea
The structural diversity of proteins makes them highly attractive as self-assembling bio-bricks or 'tectons' for the fabrication of biocompatible materials. In the current study, ring-forming Lsm proteins were exploited to construct novel nanostructures via supramolecular engineering. The Lsm family of proteins comprise the core of the ribonucleoprotein (RNP) complexes, crucial to RNA metabolism. They assemble into oligomeric rings of six to eight protein chains in vitro to generate an RNA-binding scaffold. Synthetic ring tectons composed of individual or fused Lsm sequences from S.cerevisiae were engineered so as to self assemble into simplified ring structures. My work focused on two polyprotein forms, fusions of Lsm[4+1] and Lsm[2+3]. In solution these recombinant Lsm polyproteins self-organise into robust string structures (7.5 nm x 5.0 nm), that maintain their intrinsic RNA-binding ability. Elevated levels of overlaid ring pairs are favoured at low ionic strength, indicating electrostatic mediated packing of Lsm rings. SAXS output reveals these ring pairs to be assembled in a staggered conformation rather than coaxially stacked. Using chemical modification, Lsm tectons were further fabricated into new coherent architectures. Cys residues engineered into two opposing tecton faces promoted covalent interactions between rings, generating disulfide-bonded Lsm[4+1]₄ clusters (11-12 nm) that were readily disengaged by reduction agents. This cluster coordination, resulting in organisations that are sensitive to EDTA, salt concentration and pH. A Ni²⁺-chelated Lsm[4+1]₄ cluster displayed structural similarities to its Cys-linked counterpart; this four-ringed assembly possessing an increased stability compared to Lsm[4+1]₄, although RNA-binding was compromised. In the presence of Cu²⁺ or Co²⁺ the tecton Lsm3₈ organised as ring pairs that were coaxially organised, so displaying potential as a precursor to an Lsm-based tubule. This thesis also describes crystallisation progress towards and atomic-resolution structure of the Lsm[4+1]₄ tecton. With highly pure samples, screening and optimisation across a range of conditions and crstallisation avenues allowed collection of six native datasets (at ̃ 3 Å). This data could serve to solve the first crystal structure of an Lsm polyprotein ring at high resolution. The use of Lsm rings to construct new and functional nanostructures is still in its infancy. However, this project illustrates their potential as robust and highly stable tectons, suitable for engineering endeavours. The construction of Lsm tectons into controllable higher-order superstructures, discussed in this thesis, may have future applications in RNA housing and delivery capsules or as next-generation bio-inspired nanosensors.