Fluorescence correlation spectroscopic study of serpin depolymerization by computationally designed peptides |
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Authors: | Chowdhury Pramit Wang Wei Lavender Stacey Bunagan Michelle R Klemke Jason W Tang Jia Saven Jeffrey G Cooperman Barry S Gai Feng |
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Institution: | Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA. |
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Abstract: | Members of the serine proteinase inhibitor (serpin) family play important roles in the inflammatory and coagulation cascades. Interaction of a serpin with its target proteinase induces a large conformational change, resulting in insertion of its reactive center loop (RCL) into the main body of the protein as a new strand within beta-sheet A. Intermolecular insertion of the RCL of one serpin molecule into the beta-sheet A of another leads to polymerization, a widespread phenomenon associated with a general class of diseases known as serpinopathies. Small peptides are known to modulate the polymerization process by binding within beta-sheet A. Here, we use fluorescence correlation spectroscopy (FCS) to probe the mechanism of peptide modulation of alpha(1)-antitrypsin (alpha(1)-AT) polymerization and depolymerization, and employ a statistical computationally-assisted design strategy (SCADS) to identify new tetrapeptides that modulate polymerization. Our results demonstrate that peptide-induced depolymerization takes place via a heterogeneous, multi-step process that begins with internal fragmentation of the polymer chain. One of the designed tetrapeptides is the most potent antitrypsin depolymerizer yet found. |
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Keywords: | RCL reactive center loop α1-AT α1-antitrypsin FCS fluorescence correlation spectroscopy SCADS statistical computationally assisted design strategy TMR tetramethylrhodamine |
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