Proof of principle for epitope-focused vaccine design.

Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Several 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, 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 the research and development of a human respiratory syncytial virus 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 human immunodeficiency virus and influenza.

Single molecule fluorescence studies of surface-adsorbed fibronectin.

The conformation of the extracellular matrix protein fibronectin plays a critical role in regulating cell function, including cell adhesion and migration. While average conformations of large ensembles of adhesion proteins have been previously measured, cells may sensitively respond to conformational outliers. We therefore applied both single molecule imaging and spectroscopy techniques to map a range of conformational states of individual fibronectin molecules adsorbed to glass, as well as to measure their conformational fluctuations in time. Single-step photobleaching experiments confirmed single molecule sensitivity. Single molecule spectra showed fluctuations in the peak wavelength, both over a spatial ensemble of molecules and in a single molecule over time, most likely indicating the different conformational states fibronectin can assume upon surface adsorption. Single-pair fluorescence resonance energy transfer (FRET) revealed that a fraction of fibronectin molecules existed in conformations that allowed for energy transfer between the labeled cysteine residues of the two dimeric arms folded upon each other, and that fluctuations occurred in the FRET efficiency. Fluorescence polarization experiments identified two possible sources of FRET fluctuations: changes in fluorophore orientation and conformational fluctuations of fibronectin over a time scale of seconds.

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.

Fibronectin in aging extracellular matrix fibrils is progressively unfolded by cells and elicits an enhanced rigidity response.

While the mechanical properties of a substrate or engineered scaffold can govern numerous aspects of cell behavior, cells quickly start to assemble their own matrix and will ultimately respond to their self-made extracellular matrix (ECM) microenvironments. Using fluorescence resonance energy transfer (FRET), we detected major changes in the conformation of a constituent ECM protein, fibronectin (Fn), as cells fabricated a thick three-dimensional (3D) matrix over the course of three days. These data provide the first evidence that matrix maturation occurs and that aging is associated with increased stretching of fibronectin fibrils, which leads to at least partial unfolding of the secondary structure of individual protein modules. A comparison of the conformations of Fn in these 3D matrices with those constructed by cells on rigid and flexible polyacrylamide surfaces suggests that cells in maturing matrices experience a microenviroment of gradually increasing rigidity. In addition, further matrix stiffening is caused by active Fn fiber alignment parallel to the contractile axis of the elongated fibroblasts, a cell-driven effect previously described for other fibrillar matrices. The fibroblasts, therefore, not only cause matrix unfolding, but reciprocally respond to the altered Fn matrix properties by up-regulating their own rigidity response. Consequently, our data demonstrate for the first time that a matured and aged matrix has distinctly different physical and biochemical properties compared to a newly assembled matrix. This might allow cells to specifically recognise the age of a matrix.

SPARC regulates extracellular matrix organization through its modulation of integrin-linked kinase activity.

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.

The tissue engineeting puzzle: a molecular perspective.

The inability of biomaterial scaffolds to functionally integrate into surrounding tissue is one of the major roadblocks to developing new biomaterials and tissue-engineering scaffolds. Despite considerable advances, current approaches to engineering cell-surface interactions fall short in mimicking the complexity of signals through which surrounding tissue regulates cell behavior. Cells adhere and interact with their extracellular environment via integrins, and their ability to activate associated downstream signaling pathways depends on the character of adhesion complexes formed between cells and their extracellular matrix. In particular, alpha5beta1 and alphavbeta3 integrins are central to regulating downstream events, including cell survival and cell-cycle progression. In contrast to previous findings that alphavbeta3 integrins promote angiogenesis, recent evidence argues that alphavbeta3 integrins may act as negative regulators of proangiogenic integrins such as alpha5beta1. This suggests that fibronectin is critical for scaffold vascularization because it is the only mammalian adhesion protein that binds and activates alpha5beta1 integrins. Cells are furthermore capable of stretching fibronectin matrices such that the protein partially unfolds, and recent computational simulations provide structural models of how mechanical stretching affects fibronectin function. We propose a model whereby excessive tension generated by cells in contact to biomaterials may in fact render fibronectin fibrils nonangiogenic and potentially inhibit vascularization. The model could explain why current biomaterials independent of their surface chemistries and textures fail to vascularize.

Fibronectin extension and unfolding within cell matrix fibrils controlled by cytoskeletal tension.

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 FnIII(7) and FnIII(15) 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.