Single-molecule force spectroscopy with the atomic force microscope provides molecular level insights into protein function, allowing researchers to reconstruct energy landscapes and understand functional mechanisms in biology. With steadily advancing methods, this technique has greatly accelerated our understanding of force transduction, mechanical deformation, and mechanostability within single-and multi-domain polyproteins, and receptor-ligand complexes. In this focused review, we summarize the state of the art in terms of methodology and highlight recent methodological improvements for AFM-SMFS experiments, including developments in surface chemistry, considerations for protein engineering, as well as theory and algorithms for data analysis. We hope that by condensing and disseminating these methods, they can assist the community in improving data yield, reliability, and throughput and thereby enhance the information that researchers can extract from such experiments. These leading edge methods for AFM-SMFS will serve as a groundwork for researchers cognizant of its current limitations who seek to improve the technique in the future for in-depth studies of molecular biomechanics.
Although Dopa-Fe 3+ complexation is known to play an important role in mussel adhesion for providing mechanical properties, its function at the plaque/substrate interface, where actual surface adhesion occurs, remains unknown, with regard to interfacial mussel adhesive proteins (MAPs) type 3 fast variant (fp-3F) and type 5 (fp-5). Here, we confirmed Dopa-Fe 3+ complexation of interfacial MAPs and investigated the effects of Dopa-Fe 3+ complexation regarding both surface adhesion and cohesion. The force measurements using surface forces apparatus (SFA) analysis showed that intrinsic strong surface adhesion at low pH, which is similar to the local acidified environment present during the secretion of adhesive proteins, vanishes by Dopa-Fe 3+ complexation and alternatively, strong cohesion is generated in higher pH conditions similar to seawater. A high Dopa content increased the capacity for both surface adhesion and cohesion, but not at the same time. In contrast, a lack of Dopa resulted in both weak surface adhesion and cohesion without significant effects of Fe 3+ complexation. Our findings shed light on how mussels regulate Dopa functionality at the plaque/substrate interface, in response to the microenvironment, and might provide new insight for the design of mussel-inspired biomaterials.
Coacervation of mussel adhesive proteins (MAPs) is proposed as a potential strategy that mussels may use during secretion due to their high concentration density, lack of dispersion into seawater, and low interfacial tension. Particularly, coacervations of interfacial MAPs, foot protein type‐3 fast variant (fp‐3F) and type‐5 (fp‐5), are important in the initial mussel adhesion process due to the relationship between the easy secretion/surface wetting properties of the coacervate and primer‐like surface adhesive role of interfacial MAPs, which directly contact the marine surface. To the best of the authors' knowledge, this is the first report on coacervate formation of major recombinant interfacial MAPs with high charge densities and the highest 3,4‐dihydroxyphenylalanine (Dopa) contents. Specifically, salt‐induced coacervation of fp‐3F is observed at low pH values corresponding to the acidified environment of the distal depression during mussel secretion. In addition, it shows enthalpy driven upper critical solution temperature behavior, possibly relying on bridging interactions between like‐charged cationic fp‐3Fs including salt‐bridge and cation–π/π–π interactions in the presence of specific counterions, supported by Raman spectroscopy. It is believed that this study has broadened the scope of the understanding of coacervation of MAPs and may provide new insight for responsive biomaterial design.
Misaminoacylation of 3,4-dihydroxyphenylalanine (Dopa) molecules to tRNA(Tyr) by endogenous tyrosyl-tRNA synthetase allowed the quantitative replacement of tyrosine residues with a yield of over 90 % by an in vivo residue-specific incorporation strategy, to create, for the first time, engineered mussel adhesive proteins (MAPs) in Escherichia coli with a very high Dopa content, close to that of natural MAPs. The Dopa-incorporated MAPs exhibited a superior surface adhesion and water resistance ability by assistance of Dopa-mediated interactions including the oxidative Dopa cross-linking, and furthermore, showed underwater adhesive properties comparable to those of natural MAPs. These results propose promising use of Dopa-incorporated engineered MAPs as bioglues or adhesive hydrogels for practical underwater applications.
Complex coacervates are a dense liquid phase of oppositely charged polyions formed by the associative separation of a mixture of polyions. Coacervates have been widely employed in many fields including the pharmaceutical, cosmetic, and food industries due to their intriguing interfacial and bulk material properties. More recently, attempts to develop an effective underwater adhesive have been made using complex coacervates that are based on recombinant mussel adhesive proteins (MAPs) due to the water immiscibility of complex coacervates and the adhesiveness of MAPs. MAP-based complex coacervates contribute to our understanding of the physical nature of complex coacervates and they provide a promising alternative to conventional invasive surgical repairs. Here, this review provides an overview of recombinant MAP-based complex coacervations, with an emphasis on their characterization and the uses of such materials for applications in the fields of biomedicine and tissue engineering.
We used single-molecule
AFM force spectroscopy (AFM-SMFS) in combination
with click chemistry to mechanically dissociate anticalin, a non-antibody
protein binding scaffold, from its target (CTLA-4), by pulling from
eight different anchor residues. We found that pulling on the anticalin
from residue 60 or 87 resulted in significantly higher rupture forces
and a decrease in k
off by 2–3 orders
of magnitude over a force range of 50–200 pN. Five of the six
internal anchor points gave rise to complexes significantly more stable
than N- or C-terminal anchor points, rupturing at up to 250 pN at
loading rates of 0.1–10 nN s–1. Anisotropic
network modeling and molecular dynamics simulations helped to explain
the geometric dependency of mechanostability. These results demonstrate
that optimization of attachment residue position on therapeutic binding
scaffolds can provide large improvements in binding strength, allowing
for mechanical affinity maturation under shear stress without mutation
of binding interface residues.
Misaminoacylation of 3,4‐dihydroxyphenylalanine (Dopa) molecules to tRNATyr by endogenous tyrosyl‐tRNA synthetase allowed the quantitative replacement of tyrosine residues with a yield of over 90 % by an in vivo residue‐specific incorporation strategy, to create, for the first time, engineered mussel adhesive proteins (MAPs) in Escherichia coli with a very high Dopa content, close to that of natural MAPs. The Dopa‐incorporated MAPs exhibited a superior surface adhesion and water resistance ability by assistance of Dopa‐mediated interactions including the oxidative Dopa cross‐linking, and furthermore, showed underwater adhesive properties comparable to those of natural MAPs. These results propose promising use of Dopa‐incorporated engineered MAPs as bioglues or adhesive hydrogels for practical underwater applications.
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