Substrate Partitioning into Protein Macromolecular Frameworks for Enhanced Catalytic Turnover

Abstract

Spatial partitioning of chemical processes is an important attribute of many biological systems, the effect of which is reflected in the high efficiency of enzymes found within otherwise chaotic cellular environments. Barriers, often provided through the formation of compartments or phase segregation, gate the access of macromolecules and small molecules within the cell and provide an added level of metabolic control. Taking inspiration from nature, we have designed virus-like particles (VLPs) as nanoreactor compartments that sequester enzyme catalysts and have used these as building blocks to construct 3D protein macromolecular framework (PMF) materials, which are structurally characterized using small-angle X-ray scattering (SAXS). The highly charged PMFs form a separate phase in suspension, and by tuning the ionic strength, we show positively charged molecules preferentially partition into the PMF, while negatively charged molecules are excluded. This molecular partitioning was exploited to tune the catalytic activity of enzymes enclosed within the individual particles in the PMF, the results of which showed that positively charged substrates had turnover rates that were 8500× faster than their negatively charged counterparts. Moreover, the catalytic PMF led to cooperative behavior resulting in charge dependent trends opposite to those observed with individual P22 nanoreactor particles.

Bioinspired Approaches to Self-Assembly of Virus-like Particles: From Molecules to Materials

“…47 The templating G6 dendrimer can subsequently be removed, under high ionic strength conditions, leaving the overall charge on the framework substantially negative. 4 The resultant material maintains the ordered FCC lattice structure of the P22 VLPs and is highly robust due to the ditopic Dec-Dec interparticle connectivity. These PMFs remain assembled after heating, freezing, drying, and transfer into organic solvents.…”

“…The development of synthetic nanomaterials based on P22 VLPs demonstrates how the potential for hierarchical self-assembly can be applied to other self-assembling capsid structures across multiple length scales toward future bioinspired functional materials. 4 A catalytic material with enhanced catalytic eff iciency and substrate selectivity was developed based on the collective behavior of a higherorder assembly of P22 VLPs.…”

Rapid design and prototyping of biocatalytic virus-like particle nanoreactors

“…Encapsulation of enzymes by heterologous protein cages such as VLPs can improve resistance to proteases, chaotropes, and thermal denaturation [5][6][7][8]. These protective properties, combined with the flexibility to encapsulate multiple enzymes [9][10][11], have increased interest in VLP engineering as nanocompartments for applied in vitro [12,13] and in vivo [7,8,14,15] biocatalysis in manufacturing and health.…”

“…By fusing a protein cargo to the SP, or simply to the portion of SP that interacts with the inner surface of the capsid [20], the fusion partner is encapsulated during capsid assembly [16]. P22 VLPs offer great potential for co-localisation of small metabolic pathways [9] and biocatalytic nanoreactors assembled from P22 VLPs have been engineered to enable the production of molecular hydrogen [12]; and developed as hierarchical, responsive biomaterials with enhanced catalytic properties [13,21].…”

Simultaneous Organic and Inorganic Host‐Guest Chemistry within Pillararene‐Protein Cage Frameworks

“… [24] Due to their uniform shape and size, both cages, that is, Ft and aFt have been ideal candidates to be organized into multifunctional higher‐order superlattices. They have already been used to create higher‐order crystalline structures by self‐assembly, [25] together with organic dyes, [26] dendrons and dendrimers,[ 27 , 28 , 29 ] polypeptides and proteins,[ 25 , 30 , 31 ] metal ions or nanoparticles,[ 32 , 33 , 34 , 35 ] and macrocyclic molecules [36] with varying potential applications in catalysis,[ 31 , 37 ] storage, and separation. Even though proteins are known for their relatively low stability outside aqueous media, the structural robustness of these materials in dry state has been previously demonstrated.…”