RESUMEN
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.
Asunto(s)
Péptidos , Estructura Cuaternaria de Proteína , Serpinas , Espectrometría de Fluorescencia/métodos , alfa 1-Antitripsina , Secuencia de Aminoácidos , Modelos Moleculares , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Serpinas/química , Serpinas/metabolismo , alfa 1-Antitripsina/química , alfa 1-Antitripsina/metabolismoRESUMEN
Alpha(1)-antitrypsin (AT) is the most abundantly circulating human proteinase inhibitor in the serpin family. The polymerization of AT, leading to alpha(1)-antitrypsin deficiency, has been studied extensively in vitro by a variety of ensemble methods. Here we report the use of fluorescence correlation spectroscopy to gain further insight into this process. Measurements of the distributions of diffusion times of polymerizing AT, carried out at 45, 50, and 55 degrees C, clearly show the existence of a kinetic lag phase, during which short oligomers are formed, prior to the formation of heterogeneous mixtures of longer polymers, and suggest that long polymers, which appear to be metastable, are produced through the condensation of shorter oligomers.
Asunto(s)
Polímeros/química , Polímeros/metabolismo , alfa 1-Antitripsina/química , alfa 1-Antitripsina/metabolismo , Cisteína/genética , Difusión , Electroforesis en Gel de Poliacrilamida , Humanos , Microscopía Confocal/métodos , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Desnaturalización Proteica/genética , Serina/genética , Espectrometría de Fluorescencia/métodos , Temperatura , Factores de Tiempo , alfa 1-Antitripsina/genéticaRESUMEN
The helix-coil transition kinetics of an alpha-helical peptide were investigated by time-resolved infrared spectroscopy coupled with laser-induced temperature-jump initiation method. Specific isotope labeling of the amide carbonyl groups with 13C at selected residues was used to obtain site-specific information. The relaxation kinetics following a temperature jump, obtained by probing the amide I' band of the peptide backbone, exhibit nonexponential behavior and are sensitive to both initial and final temperatures. These data are consistent with a conformation diffusion process on the folding energy landscape, in accord with a recent molecular dynamics simulation study.