Stretched Extracellular Matrix Proteins Turn Fouling and Are Functionally Rescued by the Chaperones Albumin and Casein William C. Little,+ Ruth Schwartlander,+ Michael L. Smith,# Delphine Gourdon, and Viola Vogel* Department of Materials, ETH Zurich, CH-8093 Zurich, Switzerland Received July 23, 2009 NANO LETTERS 2009 Vol. 9, No. 12 4158-4167 ABSTRACT While evidence is mounting that cells exploit protein unfolding for mechanochemical signal conversion (mechanotransduction), what mechanisms are in place to deal with the unwanted consequences of exposing hydrophobic residues upon force-induced protein unfolding? Here, we show that mechanical chaperones exist that can transiently bind to hydrophobic residues that are freshly exposed by mechanical force. The stretch-upregulated binding of albumin or casein to ﬁbronectin ﬁbers is reversible and does not inhibit ﬁber contraction once the tension is released. A decade after the birth of single-molecule mechanics,1-3 it is well established that mechanical forces can unfold proteins, and the unfolding trajectories of many proteins have been experimentally mapped or simulated (as reviewed in refs 4-7). Proteins can refold reversibly once the force is sufﬁciently reduced.8 Protein unfolding is physiologically signiﬁcant, and cell-generated tensile forces can unfold extracellular9-11 as well as intracellular12,13 proteins. On the extracellular side, cell-generated forces are sufﬁcient to stretch and then unfold ﬁbronectin (Fn) ﬁbers.9-11 Some experimental evidence14 and many predictions15,16 have shown that forced unfolding may alter binding sites or expose otherwise cryptic molecular recognition sites of extracellular matrix (ECM) proteins. On the intracellular side, force- induced unfolding of p130cas can expose phosphorylation sites.13 Mechanical stress applied by living cells to their surroundings ranges from 1 up to 10 nN per µ m2.17-20 While it is increasingly recognized that force-induced protein unfolding is an important motif by which cells can sense and respond to mechanical forces (mechanotransduc- tion processes), protein unfolding may also have deleterious consequences. As for chemically or thermally denatured proteins, the mechanical exposure of hydrophobic residues should also lead to nonspeciﬁc protein adsorption, aggrega- * To whom correspondence should be addressed. Address: Laboratory of Biologically Oriented Materials, Department of Materials, Wolfgang-Pauli- Strasse 10, HCI F 443, ETH Zurich, CH-8093 Zurich, Switzerland. Phone: +41 44 632 0887. Fax: +41 44 632 10 73. E-mail: viola.vogel@mat.ethz.ch. # Current address: Boston University, Department of Biomedical Engi- neering, Boston, MA 02215. + Authors contributed equally. 10.1021/nl902365z CCC: $40.75 Published on Web 09/10/2009  2009 American Chemical Society tion, and other processes that might be unwanted in a cellular environment. Mechanically stretched and unfolded proteins as well as protein ﬁbers should thus turn fouling. Signiﬁcantly though, the necessity of chaperones that transiently rescue the functions of mechanically unfolded proteins by inhibiting uncontrolled nonspeciﬁc binding has never been discussed before. In order to block nonspeciﬁc protein binding and to prevent loss of protein function when in contact with hydrophobic surfaces, many molecules were developed by the biomaterials community to render surfaces nonfouling.21 Biological analogues that can turn surfaces nonfouling are albumin and casein, which found widespread technical applications. Albumin, which comprises ∼55% of proteins in serum, is highly concentrated in the extracellular environment.22-24 Its three homologous domains consist of two helical subdomains that in total provide a variety of binding sites for both hydrophobic and negatively charged hydrophilic moieties. Casein, comprising ∼75% of the protein found in mammalian milk, has a highly ﬂexible amino acid chain with a large number of hydrophobic residues exposed due to its abundant proline residues.25 To date, however, nothing is known about the binding behavior of albumin and casein to proteins that are mechanically stretched and unfolded. Albumin and casein have both been characterized as extracellular chaperones, after it was shown that they could antagonize aggregation of heat-denatured proteins and assist in their functional recovery,22,24,26,27 The objective of this study was to ask, ﬁrst of all, whether the stretching of Fn ﬁbers turns them fouling and, subse-