A Study in Semenogelin I Hydrogel Aggregation Kinetics

In earlier investigations, we have shown that a peptide sequence, Sg I 38-48, derived from the protein human semenogelin I forms a hydrogel. In this work, we describe approaches to examine the kinetics of aggregation of both Sg I 38-48 and an extended sequence, Sg I 37-49. The kinetics experiments are based on using the chromophore Thioflavin T. When bound to extended β-sheet fibrils the intensity of Thioflavin T fluorescence increases dramatically and this can be used to monitor fibril formation. The fibrils have been shown previously by CD and IR spectroscopy to consist of extended β-sheets. In the kinetics experiments, the peptide is first isolated as a monomer employing gel filtration under acidic conditions when it does not aggregate. The pH is then adjusted to pH 8 to trigger fibril formation and the kinetics of aggregation is monitored using a plate reader, with an excitation and an emission wavelength of 440 nm and 480 nm, respectively. We have studied the effect of concentration, pH, and salt on the kinetics of aggregation. We have also carried out disc centrifugation studies in order to determine the size and homogeneity of the aggregates formed. The next step will be to fit our data to a mechanism. For this purpose we are using Global Fitter, and our preliminary data suggest an initial nucleation step from which the fibril extends. We have also carried out rheology experiments in order to characterize the behavior of the Sg I 38-48 sequence, which have confirmed that this peptide forms a hydrogel. These have also shown that the gel is able to recover its original strength after a strain sweep has broken the gel.