Synthetic hydrogels have been molecularly engineered to mimic the invasive characteristics of native provisional extracellular matrices: a combination of integrin-binding sites and substrates for matrix metalloproteinases (MMP) was required to render the networks degradable and invasive by cells via cell-secreted MMPs. Degradation of gels was engineered starting from a characterization of the degradation kinetics (kcat and Km) of synthetic MMP substrates in the soluble form and after crosslinking into a 3D hydrogel network. Primary human fibroblasts were demonstrated to proteolytically invade these networks, a process that depended on MMP substrate activity, adhesion ligand concentration, and network crosslinking density. Gels used to deliver recombinant human bone morphogenetic protein-2 to the site of critical defects in rat cranium were completely infiltrated by cells and remodeled into bony tissue within 4 wk at a dose of 5 g per defect. Bone regeneration was also shown to depend on the proteolytic sensitivity of the matrices. These hydrogels may be useful in tissue engineering and cell biology as alternatives for naturally occurring extracellular matrix-derived materials such as fibrin or collagen. extracellular matrix ͉ biomaterials ͉ proteolytic degradation A dvances in the field of tissue engineering are connected to the performance of biomaterials that help in guiding tissue formation or regeneration. Design principles in biomaterials have typically been influenced by the function of the extracellular matrix (ECM). Because the ECM has been demonstrated to play a key role in signal transduction (1-3), the development of materials that can specifically and molecularly interact with cells has become an emerging area of research (4, 5). The regulation of cell behavior through receptor-mediated adhesion [e.g., by functionalizing materials with integrin-binding oligopeptides (6, 7)] and by growth factors (8) has thus been targeted.We and others have been focusing on the development of synthetic materials that are targeted to assist tissue regeneration (9-11). Placed at the site of a defect, such materials should actively and temporarily participate in the regeneration process by providing a platform on which cell-triggered remodeling could occur. Consequently, these matrices must display some key characteristics of the provisional ECM. One of the critical initial functions of the fibrin-rich network that fills tissue defects after trauma, almost regardless of the injury site, lies in its ability to foster the invasion of inflammatory cells. These cells then initiate the remodeling process by partially degrading the matrix and by secreting molecular signals for attraction and differentiation of other cell types such as fibroblasts that build up new ECM (12). Because the provisional ECM presents itself in such situations often as a biophysical barrier to these cells, invasion and remodeling depend on the action of cell-secreted proteases enabling cell migration by clearing of a path (13,14). Thereby, matrix met...

9‐Fluorenylmethoxycarbonyl (Fmoc) amino acids were first used for solid phase peptide synthesis a little more than a decade ago. Since that time, Fmoc solid phase peptide synthesis methodology has been greatly enhanced by the introduction of a variety of solid supports, linkages, and side chain protecting groups, as well as by increased understanding of solvation conditions. These advances have led to many impressive syntheses, such as those of biologically active and isotopically labeled peptides and small proteins. The great variety of conditions under which Fmoc solid phase peptide synthesis may be carried out represents a truly “orthogonal” scheme, and thus offers many unique opportunities for bioorganic chemistry.

Breakdown of triple-helical interstitial collagens is essential in embryonic development, organ morphogenesis and tissue remodelling and repair. Aberrant collagenolysis may result in diseases such as arthritis, cancer, atherosclerosis, aneurysm and fibrosis. In vertebrates, it is initiated by collagenases belonging to the matrix metalloproteinase (MMP) family. The three-dimensional structure of a prototypic collagenase, MMP-1, indicates that the substrate-binding site of the enzyme is too narrow to accommodate triple-helical collagen. Here we report that collagenases bind and locally unwind the triple-helical structure before hydrolyzing the peptide bonds. Mutation of the catalytically essential residue Glu200 of MMP-1 to Ala resulted in a catalytically inactive enzyme, but in its presence noncollagenolytic proteinases digested collagen into typical 3/4 and 1/4 fragments, indicating that the MMP-1(E200A) mutant unwinds the triple-helical collagen. The study also shows that MMP-1 preferentially interacts with the alpha2(I) chain of type I collagen and cleaves the three alpha chains in succession. Our results throw light on the basic mechanisms that control a wide range of biological and pathological processes associated with tissue remodelling.

Peptide-amphiphiles with collagen-model head groups and dialkyl chain tails have been synthesized and shown to self-assemble into highly ordered polyPro II like triple-helical structures when dissolved in aqueous subphases. Evidence for this self-assembly process has been obtained from (a) compression of stable peptide-amphiphile monolayers to molecular areas comparable with triple-helical areas, (b) circular dichroism spectra and melting curves characteristic of triple-helices, and (c) two-dimensional NMR spectra indicative of stable triple-helical structure at low temperatures and melted triple-helices at high temperatures. The thermal stability of the collagen-like structure in the peptide-amphiphile is substantially higher (ΔT m = 15−20 °C) than that of peptides without lipidation. The assembly process driven by the hydrophobic tail may provide a general method for creating well-defined protein molecular architecture using a minimalist peptide-based approach.

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Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme–inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site.

The proteolysis of collagen triple-helical structure (collagenolysis) is a poorly understood yet critical physiological process. Presently, matrix metalloproteinase 1 (MMP-1) and collagen triple-helical peptide models have been utilized to characterize the events and calculate the energetics of collagenolysis via NMR spectroscopic analysis of 12 enzyme-substrate complexes. The triple-helix is bound initially by the MMP-1 hemopexin-like (HPX) domain via a four amino acid stretch (analogous to type I collagen residues 782–785). The triple-helix is then presented to the MMP-1 catalytic (CAT) domain in a distinct orientation. The HPX and CAT domains are rotated with respect to one another compared with the X-ray “closed” conformation of MMP-1. Back-rotation of the CAT and HPX domains to the X-ray closed conformation releases one chain out of the triple-helix, and this chain is properly positioned in the CAT domain active site for subsequent hydrolysis. The aforementioned steps provide a detailed, experimentally-derived, and energetically favorable collagenolytic mechanism, as well as significant insight into the roles of distinct domains in extracellular protease function.

Peptide-amphiphiles with collagen-model head groups and dialkyl chain tails have been shown previously to self-assemble into highly ordered polyPro II-like triple-helical structures when dissolved in aqueous subphases. In the present study, we have examined peptide-amphiphiles containing monoalkyl chain tails for similar self-assembly behaviors. The structure of a collagen-model peptide has been characterized with and without an N-terminal hexanoic acid (C 6 ) modification. Evidence for a self-assembly process of both the peptide and peptide-amphiphile has been obtained from (a) circular dichroism spectra and melting curves characteristic of triple-helices, (b) one-dimensional NMR spectra indicative of stable triple-helical structure at low temperatures and melted triple helices at high temperatures, and (c) pulsed-field gradient NMR experiments demonstrating different self-diffusion coefficients between proposed triple-helical and non-triple-helical species. The peptide-amphiphile appeared to form monomeric triple helices. The thermal stability of the collagen-like structure in the peptide-amphiphile was found to increase as the monoalkyl tail chain length is increased over a range of C 6 to C 16 . The assembly process driven by the hydrophobic tail, albeit monoalkyl or dialkyl, may provide a general method for creating well-defined protein molecular architecture. Peptide-amphiphile structures possessing these alkyl moieties have the potential to be used for biomaterial surface modification to improve biocompatibility or, by mimicing fusion of viral envelopes with cellular membranes, as drug delivery vehicles.