SoftComp webinar: Prof. Randall Kemian – 20 February 2025, 15:00 CET
5th February 2025
Fig. 2. Schematic illustration of the blending of humins and PLA, the production of humins from sugar-based feedstocks, and the production of PLA from sugar cane crop residues. Copyright: Dilhan Kandemir. All rights reserved.
Upcycling Humins into Valuable Materials
10th February 2025

Impact of Aspect Ratio on Protein Network Assembly

Interconnected networks of high aspect ratio (AR) bio-polymers are ubiquitous in nature. A research team from SoftComp partner Leeds and Oxfordshire has now shown why high AR bio-polymer building blocks confer significant functional advantages. These include; increased mechanical strength at equivalent protein concentrations; and the rapid assembly of homogenous networks, above a critical concentration, crucial for in vivo biological processes e.g. blood clotting. In addition to uncovering the function advantages of filamentous proteins for hydrogel formation in vivo, manipulating AR also provides a novel parameter in the design of new biomaterials.

To shed light on the advantages of high aspect biopolymers as network building blocks, the scientists engineered proteinaceous building blocks with varying numbers of protein L (pL) domains, creating seven building blocks with ARs from one to seven. Using shear rheology and small-angle neutron scattering (SANS) to characterise the mechanical and structural properties of photochemically crosslinked pL networks at different volume fractions, φ, they showed that AR is a crucial property that defines network architecture and mechanics. Networks constructed from higher AR building blocks exhibit more homogeneous structures and higher storage moduli due to a shift from translational diffusion limited (TDL) to rotationally diffusion limited (RDL) network formation because building blocks with increased AR lack the space to rotate freely (Figure 1). The transition between these regimes is modelled by the geometric free rotation limit of a rigid cylindrical rod, also known as the transition from the dilute to the semi-dilute regime for rod-like particles.

Formation kinetics lag time, τ, measurements (supplemented with dynamic light scattering) of pL polyproteins, as a function of volume fraction, further demonstrate the presence of the TDL to RDL transition (Fig. 2a,b). Where the fitted crossover volume fraction, φcrit, extracted using a mean free path based empirical fit (Fig. 2 caption), matches the values determined by a geometric free rotation limit model (Eqn 1). Finally, for comparison, the scientists studied a fibrin network (Fig. 2c,d) and observe the same transition from TDL to RDL formation, confirming that living systems exploit AR for their network assembly.

 

Read more:
Hughes M. D. G. et al., Nat. Commun. 14, 5593 (2023)

SoftComp partner:
University of Leeds

Figure 1: Aspect ratio is a crucial network building block property, defining network formation, architecture, and mechanics in synthetic and living systems. (Left) Schematic representation of the aspect ratios (AR) of protein L polyprotein constructs and fibrin proto-fibers. (Middle) A schematic representation of the two dominated formation regimes: translation diffusion limited (TDL) where building blocks can only interact by translating their centre of mass; and rotationally diffusion limited (RDL) where building blocks are able to interact purely via rotation about their centre of mass. (Right) The fitted increase in mechanical rigidity as a function of aspect ratio with inserted schematics showing the change in structural topology as the system transitions from TDL to RDL formation above the critical aspect ratio, ARc. Copyright: Image partly published in Hughes M. D. G. et al., Nat. Commun. 14, 5593 (2023) under a Creative Commons Attribution 4.0 International License.
Figure 1: Aspect ratio is a crucial network building block property, defining network formation, architecture, and mechanics in synthetic and living systems. (Left) Schematic representation of the aspect ratios (AR) of protein L polyprotein constructs and fibrin proto-fibers. (Middle) A schematic representation of the two dominated formation regimes: translation diffusion limited (TDL) where building blocks can only interact by translating their centre of mass; and rotationally diffusion limited (RDL) where building blocks are able to interact purely via rotation about their centre of mass. (Right) The fitted increase in mechanical rigidity as a function of aspect ratio with inserted schematics showing the change in structural topology as the system transitions from TDL to RDL formation above the critical aspect ratio, ARc. Copyright: Image partly published in Hughes M. D. G. et al., Nat. Commun. 14, 5593 (2023) under a Creative Commons Attribution 4.0 International License.
Figure 2: Increasing aspect ratio (AR) shifts the dominant network formation mechanism of synthetic (pL hydrogels) and natural (fibrin) protein networks from translational to rotational diffusion. a) Schematic representation of pL7 building block which has an aspect ratio of 7, with inset of pL7 structure as predicted by AlphaFold. b) Network formation lag time, τ, extracted from gelation curves as a function of protein volume fraction for pL7 hydrogel networks. c) Schematic representation of fibrin protofibrils which have an approximate aspect ratio of 44 (20-25 monomer units), with inset of the crystal structure of human fibrinogen (PDB code: 3GHG) arranged in an offset stacked structure. d) Network formation lag time, τ, extracted from gelation curves as a function of protein volume fraction for fibrin networks. Solid lines (in panels b) and d)) show the fits using τ=τ_t/ϕ+τ_r, while dotted lines show the extracted values for the crossover volume fraction i.e. when τ_t/ϕ_crit =τ_r (equivalent fibrin concentration of 0.7 ± 0.1 mg∙ml-1). Additionally, the predicted values from Eqn. 1 are shown for comparison. The grey region in panel d) shows the range of fibrinogen volume fractions below 0.75 mg∙ml-1 corresponding to critically low blood fibrinogen concentrations i.e., hypofibrinogenemia. Copyright: Image published in Hughes M. D. G. et al., Nat. Commun. 14, 5593 (2023) under a Creative Commons Attribution 4.0 International License.
Figure 2: Increasing aspect ratio (AR) shifts the dominant network formation mechanism of synthetic (pL hydrogels) and natural (fibrin) protein networks from translational to rotational diffusion. a) Schematic representation of pL7 building block which has an aspect ratio of 7, with inset of pL7 structure as predicted by AlphaFold. b) Network formation lag time, τ, extracted from gelation curves as a function of protein volume fraction for pL7 hydrogel networks. c) Schematic representation of fibrin protofibrils which have an approximate aspect ratio of 44 (20-25 monomer units), with inset of the crystal structure of human fibrinogen (PDB code: 3GHG) arranged in an offset stacked structure. d) Network formation lag time, τ, extracted from gelation curves as a function of protein volume fraction for fibrin networks. Solid lines (in panels b) and d)) show the fits using τ=τ_t/ϕ+τ_r, while dotted lines show the extracted values for the crossover volume fraction i.e. when τ_t/ϕ_crit =τ_r (equivalent fibrin concentration of 0.7 ± 0.1 mg∙ml-1). Additionally, the predicted values from Eqn. 1 are shown for comparison. The grey region in panel d) shows the range of fibrinogen volume fractions below 0.75 mg∙ml-1 corresponding to critically low blood fibrinogen concentrations i.e., hypofibrinogenemia. Copyright: Image published in Hughes M. D. G. et al., Nat. Commun. 14, 5593 (2023) under a Creative Commons Attribution 4.0 International License.
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