Two-step nucleation pathways in which disordered, amorphous, or dense liquid states precede the appearance of crystalline phases have been reported for a wide range of materials, but the dynamics of such pathways are poorly understood. Moreover, whether these pathways are general features of crystallizing systems or a consequence of system-specific structural details that select for direct versus two-step processes is unknown. Using atomic force microscopy to directly observe crystallization of sequence-defined polymers, we show that crystallization pathways are indeed sequence dependent. When a short hydrophobic region is added to a sequence that directly forms crystalline particles, crystallization instead follows a two-step pathway that begins with the creation of disordered clusters of 10-20 molecules and is characterized by highly non-linear crystallization kinetics in which clusters transform into ordered structures that then enter the growth phase. The results shed new light on non-classical crystallization mechanisms and have implications for the design of self-assembling polymer systems.
Solid-binding peptides (SBPs) recognizing
inorganic and synthetic
interfaces have enabled a broad range of materials science applications
and hold promise as adhesive or morphogenetic control units that can
be genetically encoded within desirable or designed protein frameworks.
To date, the underlying relationships governing both SBP–surface
and SBP–SBP interactions and how they give rise to different
adsorption mechanisms remain unclear. Here, we combine protein engineering,
surface plasmon resonance characterization, and molecular dynamics
(MD) simulations initiated from Rosetta predictions to gain insights
on the interplay of amino acid composition, structure, self-association,
and adhesion modality in a panel of variants of the Car9 silica-binding
peptide (DSARGFKKPGKR) fused to the C-terminus of superfolder
green fluorescent protein (sfGFP). Analysis of kinetics, energetics,
and MD-predicted structures shows that the high-affinity binding of
Car9 to the silanol-rich surface of silica is dominated by electrostatic
contributions and a spectrum of several persistent interactions that,
along with a high surface population of bound molecules, promote cooperative
interactions between neighboring SBPs and higher order structure formation.
Transition from cooperative to Langmuir adhesion in sfGFP-Car9 variants
occurs in concert with a reduction of stable surface interactions
and self-association, as confirmed by atomic force microscopy imaging
of proteins exhibiting the two different binding behaviors. We discuss
the implications of these results for the de novo design of SBP–surface
binding systems.
Biomimetic silica formation, a process that is largely driven by proteins, has garnered considerable interest in recent years due to its role in the development of new biotechnologies. However, much remains unknown of the molecular-scale mechanisms underlying the binding of proteins to biomineral surfaces such as silica, or even of the key residue-level interactions between such proteins and surfaces. In this study, we employ molecular dynamics (MD) simulations to study the binding of R5-a 19-residue segment of a native silaffin peptide used for in vitro silica formation-to a silica surface. The metadynamics enhanced sampling method is used to converge the binding behavior of R5 on silica at both neutral (pH 7.5) and acidic (pH 5) conditions. The results show fundamental differences in the mechanism of binding between the two cases, providing unique insight into the pH-dependent ability of R5 and native silaffin to precipitate silica. We also study the effect of phosphorylation of serine residues in R5 on both the binding free energy to silica and the interfacial conformation of the peptide. Results indicate that phosphorylation drastically decreases the binding free energy and changes the structure of silica-adsorbed R5 through the introduction of charge and steric repulsion. New mechanistic insights from this work could inform rational design of new biomaterials and biotechnologies.
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