signiﬁcantly decreased once they were stretched beyond the onset of FnIII module unfolding (1 M GdnHCL). This phenomenon is likely explained by the disruption of L8’s binding site as the relative position between FnI9 and FnIII1 is mechanically increased. Enhanced Albumin and Casein Binding Correlates with the Force-Induced Unfolding of FnIII Modules. Since albumin and casein binding signiﬁcantly increases when the Fn ﬁbers are stretched, we asked how and whether this correlates with the force-induced unfolding of FnIII modules. Since two of the 15 FnIII modules of a Fn monomer each contain a buried cysteine residue that is only exposed upon denaturation or upon forced unfolding (on FnIII7 and FnIII15), we used an assay developed by Johnson et al.,12 where cysteine-labeling dyes were utilized to indicate the force-induced exposure of buried cysteine residues in the intracellular proteins.31 In a control, iodoacetamide was used to alkylate-exposed hydrophobic patches in different Fn starting formations.32 Using these two assays, we show that stretching of the Fn ﬁbers exposes the buried cysteines on FnIII7 and FnIII15 (Figure 2a-g) and that cysteine exposure increases with mechanical strain (Figure 2h,i), similar to recent data from our group conducted on freely suspended ﬁbers (Klotzsch et al., submitted). To investigate the low strain region, the ﬁbers were deposited (140% strain) on a prestrained silicone sheet that was then fully relaxed (0% strain). Since forces of a few µ N are needed to pull the Fn ﬁbers out of a droplet, they are prestrained to approximately 140% strain when they are deposited onto the silicone sheets (Klotzsch et al., submitted). Any possible free cysteines exposed on the now relaxed Fn ﬁbers were blocked with iodoacetamide. Afterward, the ﬁbers were stretched in increments, and the newly exposed cysteines in each incre- ment were allowed to react with maleimide dye from solution. To investigate the high strain region in a second set of experiments, the Fn ﬁbers were now deposited (140% strain) on a relaxed silicone sheet. The free cysteines were ﬁrst blocked with iodoacetamide. The ﬁbers were then stretched and stained with maleimide dye in increments until a 550% absolute ﬁber strain was reached. In a control experiment, it could be demonstrated that alkylation of the buried cysteins of Fn in a denaturing solution prior to assembling the ﬁbers and stretching them blocked all maleimide binding sites (Figure 2b-d). These data suggest that the fraction of unfolded FnIII modules increases steadily with mechanical strain. In contrast to upregulated nonspeciﬁc binding of proteins to stretched ﬁbers, stretching reduces the binding of the probe ﬂuorophore bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-dis- ulfonic acid, dipotassium salt). Bis-ANS has been used to identify structural intermediates in the thermal or chemical unfolding pathways of proteins.33-39 While bis-ANS in aqueous solution alone has a negligible ﬂuorescence, it turns ﬂuorescent when bound to hydrophobic regions of partially disrupted tertiary structures and decreases again when the secondary structure is destroyed.39,40 When nonﬁbrillar Fn is chemically denatured in solution (using GdnHCl), the bis- ANS ﬂuorescence reaches maximal values at about ∼0.8 M Nano Lett., Vol. 9, No. 12, 2009 GdnHCl and then declines upon further denaturation (Sup- porting Information, Figure S1). Taken together, these ﬁndings are in agreement with previous circular dichroism studies, indicating that these are the conditions under which Fn begins to lose secondary structure.10,27-29 As Fn ﬁbers are stretched, the bis-ANS ﬂuorescence per Fn molecule declines steadily (Figure 2j,k). This trend seen for bis-ANS is in contrast to the increased binding of albumin and casein to stretched ﬁbers (Figure 1), and in agreement with the structural model proposed here. Stretched Fn Fibers Relax within Minutes Coincident with the Release of Nonspeciﬁcally Bound Proteins. Reversibility is a central attribute that has to be met by any protein whose physiological function and functional regula- tion depends on surviving many stretch and relaxation cycles. It is thus important to ask whether albumin binding to stretched Fn ﬁbers inhibits the structural relaxation of Fn ﬁbers or whether albumin is released from the ﬁber upon ﬁber relaxation. In the presence and absence of adsorbed albumin, FRET from ﬁbrillar Fn recovers when the stretched ﬁbers are relaxed, as seen in Figure 3a. While it took about a minute to manually relax the silicone sheet in the strain device and then a few more minutes to refocus, we note that relaxing the Fn ﬁbers can indeed switch their FRET signature. The contraction of the Fn ﬁbers was accompanied by the release of the nonspeciﬁcally bound proteins (Figure 3b). Upon restretching, the IA/ID ratio changes back to the original value. Speciﬁc Fn-Fn Binding Sites on the Fibrils Are Not Blocked by Nonspeciﬁc Protein Binding. To address whether nonspeciﬁc binding of albumin might block speciﬁc binding sites on Fn ﬁbers, we probed how the exposure of the Fn-Fn binding sites depends on the mechanical strain of the ﬁbers. Fn-Fn binding sites are found on the N-terminal FnI modules and on various FnIII modules,6,41 and it has been shown previously that cell-generated forces acting on Fn are needed to induce Fn ﬁbrillogenesis either ex vivo42-44 or in cell culture.45 Images of Fn ﬁbers at various strains show indeed that soluble Fn binds more strongly to stretched (horizontal orientation) rather than to relaxed (vertical orientation) ﬁbers (Figure 4); full-length soluble Fn as well as its N-terminal 70 kDa fragment (domains FnI1-FnIII1) show the same stretch upregulated trend. The pixel-by-pixel correlations in Figure 4d show more quanti- tatively that the binding of soluble Fn-633 normalized by the number of ﬁbrillar Fn molecules increases ﬁrst gently until the ﬁbers reach 100-140% absolute strain, followed by a steep increase at higher strains (Figure 4d). In contrast, while the Fn’s 70 kDa fragment also showed an increase in binding to mechanically stretched Fn ﬁbers (Figure 4e-h), its most signiﬁcant increase occurred before the ﬁbers were stretched to 100-140% absolute strain (see also Figure S5, Supporting Information). One explanation for the enhanced binding of the 70 kDa fragment at low ﬁber strain could be that low tensions induce a conformational change within FnIII1-FnIII2, opening up a cryptic site that upregulates the binding of the 70 kDa fragment,41,45,46 or in any other mechanosensitive regions. In contrast, binding of full-length 4161