Exploiting fluorescence resonance energy transfer to probe structural changes in a macromolecule during adsorption and incorporation into a growing biomineral crystal.
The growth of natural biominerals is often tightly regulated by surface adsorption and subsequent incorporation of proteins into the crystal structure. Understanding how macromolecules intercalate into inorganic crystal lattices and how incorporation affects protein structure is crucial to learning how to engineer biomimetic materials with advanced properties, yet knowledge about the molecular-level interactions between organic guests and inorganic hosts remains sparse. Here we have used fluorescence resonance energy transfer (FRET) to probe conformational changes of a macromolecule as it adsorbs to, and becomes incorporated within, a biomineral crystal. Calcium oxalate monohydrate (COM) was used as a model due to its large size and kinetic stability under a wide range of pH values. Since the conformation of the extracellular matrix protein fibronectin (Fn) is highly sensitive to local ion concentrations, major conformational changes can be observed by FRET, as Fn senses and responds to varying local ionic conditions. When transferred from a physiological buffer to a supersaturated solution, Fn's crossed-over dimeric arms separate, indicating a weakening of the electrostatic interactions which otherwise stabilize the compact conformation of the protein. Fn returns to a more compact state when binding to the flat (-101) surface of the crystal, suggesting that Fn might sense a zone of ion depletion right at the interface of the growing crystal. As the crystal begins to grow around the absorbed protein, the dimeric Fn arms separate again, potentially driven by interactions with the newly formed charged step edges forming around it during the embedding process. FRET thus reveals for the first time how local changes in the electrostatic environment during the growth of a biomineral can cause major alterations in protein conformation. The insights derived using FRET and atomic force microscopy (AFM) could stimulate novel ways to tailor and tune the properties of organic-inorganic composites by exploiting dynamically changing electrostatic guest-host interactions.
Touryan
Lara A
LA
Center for Nanotechnology, Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.
Baneyx
Gretchen
G
Vogel
Viola
V
D000327:Adsorption; D001672:Biocompatible Materials / Q000737:chemistry*; D002129:Calcium Oxalate / Q000737:chemistry; D003460:Crystallization; D005353:Fibronectins / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D018625:Microscopy, Atomic Force; D008903:Minerals / Q000737:chemistry*; D015335:Molecular Probes
D001672:Biocompatible Materials; D005353:Fibronectins; D008903:Minerals; D015335:Molecular Probes; D002129:Calcium Oxalate
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