Summary Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Multiple major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus (RSV), that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for research and development of a human RSV vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets including antigenically highly variable pathogens such as HIV and influenza.

Evidence is emerging that mechanical stretching can alter the functional states of proteins. Fibronectin (Fn) is a large, extracellular matrix protein that is assembled by cells into elastic fibrils and subjected to contractile forces. Assembly into fibrils coincides with expression of biological recognition sites that are buried in Fn's soluble state. To investigate how supramolecular assembly of Fn into fibrillar matrix enables cells to mechanically regulate its structure, we used fluorescence resonance energy transfer (FRET) as an indicator of Fn conformation in the fibrillar matrix of NIH 3T3 fibroblasts. Fn was randomly labeled on amine residues with donor fluorophores and site-specifically labeled on cysteine residues in modules FnIII7 and FnIII15 with acceptor fluorophores. Intramolecular FRET was correlated with known structural changes of Fn in denaturing solution, then applied in cell culture as an indicator of Fn conformation within the matrix fibrils of NIH 3T3 fibroblasts. Based on the level of FRET, Fn in many fibrils was stretched by cells so that its dimer arms were extended and at least one FnIII module unfolded. When cytoskeletal tension was disrupted using cytochalasin D, FRET increased, indicating refolding of Fn within fibrils. These results suggest that cell-generated force is required to maintain Fn in partially unfolded conformations. The results support a model of Fn fibril elasticity based on unraveling and refolding of FnIII modules. We also observed variation of FRET between and along single fibrils, indicating variation in the degree of unfolding of Fn in fibrils. Molecular mechanisms by which mechanical force can alter the structure of Fn, converting tensile forces into biochemical cues, are discussed. E xtracellular matrices (ECM) are complex supramolecular assemblies that control cell signaling and behavior. While many ECM proteins have been characterized, little is known about how matrix assembly alters their structure and confers functions not present in individual proteins. Less is known about mechanisms by which cell contractile forces applied to protein assemblies regulate protein function. Many ECM proteins, such as fibronectin (Fn), laminin, and thrombospondin, are large (Ͼ100 kDa), multifunctional proteins composed of repeating, structurally defined modules often less than 100 aa each. The multimodular structure may serve as a convenient way to integrate multiple functions in one molecule. For example, some modules carry cell adhesion sites, whereas others carry sites for binding other proteins and for self-assembly. Exposure of functional sites may be controlled by the organization of such sites in extracellular matrices, and by the application of force by cells (1-3). However, the molecular mechanisms regulating ECM protein assembly and the exposure of binding sites remain unclear.Fn is an ECM protein that undergoes cell-mediated assembly into insoluble, elastic fibrils. In blood and when secreted by cells such as fibroblasts, Fn exists as a soluble dimer. The two strand...

Fluorescence resonance energy transfer (FRET) between fluorophores attached to single proteins provides a tool to study the conformation of proteins in solution and in cell culture. As a protein unfolds, nanometer-scale increases in distance between donor and acceptor fluorophores cause decreases in FRET. Here we demonstrate the application of FRET to imaging coexisting conformations of fibronectin (Fn) in cell culture. Fn is a flexible 440-kDa extracellular matrix protein, with functional sites that are regulated by unfolding events. Fn was labeled with multiple donor and acceptor fluorophores such that intramolecular FRET could be used to distinguish a range of Fn conformations. The sensitivity of FRET to unfolding was tested by progressively denaturing labeled Fn using guanidium chloride. To investigate Fn conformation changes during cell binding and matrix assembly, we added labeled Fn to the culture medium of NIH 3T3 fibroblasts. Coexisting conformations of Fn were visualized using fluorescence microscopy, and spectra from specific features were measured with an attached spectrometer. Using FRET as an indicator of Fn conformation, Fn diffusely bound to cells was in a compact state, whereas Fn in matrix fibrils was highly extended. Matrix fibrils exhibited a range of FRET that suggested some degree of unfolding of Fn's globular modules. Fn in cell-associated clusters that preceded fibril formation appeared more extended than diffuse cell-bound Fn but less extended than fibrillar Fn, suggesting that Fn undergoes extension after cell binding and before polymerization. FRET thus provides an approach to gain insight into the integrin-mediated pathway of Fn fibrillogenesis. Imaging tools that discriminate between protein conformations in cell culture are essential for insight into how changes in protein structure modulate cell signaling. Fluorescence resonance energy transfer (FRET) has emerged to fill a void in techniques that provide high-resolution structural information on proteins adsorbed to surfaces or in cell culture. FRET is the nonradiative transfer of excitation energy from a fluorescent molecule, called the donor, to another molecule, called the acceptor (1, 2). It is generally detected as an increase in acceptor emission with a concomitant decrease in donor emission. Energy transfer from donor to acceptor fluorophore decays rapidly with increasing distance (the rate is inversely proportional to the sixth power of the separation), resulting in nanometer-scale sensitivity for distances up to 10 nm. By using fluorescently labeled proteins, FRET has been applied to detect protein-protein association on cell surfaces (3, 4) as well as unfolding transitions of proteins in solution (5, 6). Here we demonstrate FRET applied to imaging coexisting protein conformations in cell culture.Fibronectin (Fn) was chosen as a model protein because it undergoes dramatic conformational changes that control its biological activity, yet the underlying molecular mechanisms are largely unknown. In blood and when secreted by c...

SPARC, a 32-kDa matricellular glycoprotein, mediates interactions between cells and their extracellular matrix, and targeted deletion of Sparc results in compromised extracellular matrix in mice. Fibronectin matrix provides provisional tissue scaffolding during development and wound healing and is essential for the stabilization of mature extracellular matrix. Herein, we report that SPARC expression does not significantly affect fibronectin-induced cell spreading but enhances fibronectin-induced stress fiber formation and cell-mediated partial unfolding of fibronectin molecules, an essential process in fibronectin matrix assembly. By phage display, we identify integrin-linked kinase as a potential binding partner of SPARC and verify the interaction by co-immunoprecipitation and colocalization in vitro. Cells lacking SPARC exhibit diminished fibronectin-induced integrin-linked kinase activation and integrin-linked kinase-dependent cell-contractile signaling. Furthermore, induced expression of SPARC in SPARC-null fibroblasts restores fibronectin-induced integrin-linked kinase activation, downstream signaling, and fibronectin unfolding. These data further confirm the function of SPARC in extracellular matrix organization and identify a novel mechanism by which SPARC regulates extracellular matrix assembly.Matricellular proteins such as SPARC function as modulators of cellextracellular matrix (ECM) 2 interactions (1, 2). SPARC is considered "antiadhesive," because it does not directly support cell attachment. Moreover, it induces focal adhesion disassembly and cell rounding when the purified protein is added to spread cells (3-5). The induction of an intermediate state of cell adhesion by SPARC (6) implies a role for SPARC in the organization of ECM. Consistent with data acquired in vitro, mice with a targeted disruption of Sparc have marked developmental abnormalities in the dermis, eye, and adipose tissue (7-9) and show accelerated closure of dermal wounds (10 -11), diminished foreign body response (12), and enhanced tumor growth (13). These aberrations have been explained, in part, by altered ECM production and assembly. Specifically, tissues from SPARC-null (SϪ/Ϫ) mice contain less collagen than those from wild-type (WT) mice, and the collagen present is less mature (7). However, the mechanism by which SPARC directs ECM assembly has not been identified.The development of mature ECM requires proper formation of an organized fibronectin (Fn) matrix. The importance of Fn in the morphogenesis and patterning of tissues is established, since Fn-null mice die during early gastrulation as a result of defective cell migration (14). Integrin-linked kinase (ILK), a serine/threonine kinase that binds to the intracellular domain of ␤1 integrin immediately adjacent to the plasma membrane and is activated by ␤1 integrins and growth factors, has been shown to control the intracellular signaling cascades that influence cellular contractile elements (25). ILK interacts directly with actin and ␣-actinin-binding proteins such as th...