Based on the goals I proposed for my project this semester, approximately 50% have been completed. Not only have I been able to isolate the hsfi1 construct with up to ~75-80% purity, but I’ve also been able to conduct 2D-correlational spectroscopy studies for a hcentrin1 mutant, specifically E105K. These FT-IR correlation spectroscopy studies have allowed me to identify which secondary structures of the protein are perturbed and to what extent based on increasing thermal perturbations exerted on the sample. Furthermore, using spectral plots of these perturbations, I’ve been able to establish the sequential order of events that take place as the protein unfolds or destabilizes with increasing temperature. By comparing the sequential order of events of this mutant and that of other hcentrin mutants studied in the lab, we are able to compare the extent of stability of all wild type and mutant hcentrin proteins, with hopes of understanding the extent of instability different mutations bring to the protein. We hope to extend our 2D-COS studies to the understanding of hcentrin-hsfi1 complex formation.
Due to a number of factors previously explained in other blog posts, it’s been very difficult to fully isolate the hsfi1 construct. Nonetheless, as we’ve begun to understand the behavior of the protein more and more, we’ve modified the conditions of purification and have been very successful in moving the project forward. We have recently designed a slightly different modification of the purification protocol that we expect will greatly increase our chances of finally isolating the construct. This modification is based on the insolubility properties the protein presents, and its surprising affinity to the chromatography column used in comparison with the affinity tag ‘’GST.’’
BioMinds Poster Abstract
Conformational stability of hcen1 (E105K) upon thermal perturbation using two-dimensional correlational spectroscopy and hsfi1 construct isolation for subsequent dynamic characterization studies of hsfi1-hcen complexes
A. Castillo Rodriguez †, B. Pastrana †‡
† University of Puerto Rico, Mayaguez Campus
‡ Protein structural characterization center, chemistry department
research area: Biophysics and biochemistry
Centrins are a low molecular weight (~2000), subfamily within the EF-hand Calmodulin superfamily that play an essential role in centrosome duplication and contraction of centrin-based fiber systems. Although elucidation of centrin function and structure is yet to be fully explained, evidence shows interaction with other protein targets such as sfi1.
Sfi1 is a large protein mainly localized in the centrosome of higher eukaryotes. Its large sequence reveals 23 amino acid repeats in human proteins, separated by 10 residue linkers. Experiments have revealed the presence of a physical interaction between hsfi1 and centrin molecules, suggesting that each of the 23 amino acid repeats present in Sfi1 comprise a centrin-binding site. Nonetheless, very little is known of sfi1 proteins, and its structure is yet to be fully elucidated.
In order to probe conformationally sensitive delocalized states of Hcen1 E105K upon thermal perturbation and defeat spectral congestion, transient conformational changes were investigated using nonlinear Fourier transform infrared IR spectroscopy of the amide I’ vibrations and amide II’ bands in the mid IR region of the spectrum. Two-dimensional correlational spectroscopy (2DCOS) reveals a transient instability in secondary structure of the mutant protein as a function of increasing temperature. Backbone correlations confirm order of events, suggesting weak conformational perturbations in the loop regions initiate alpha-helical instability of the protein, thus promoting subsequent unsteadiness of the ß-strand composition. This latter event, in turn, finally initiates instability of calcium-binding sites of the protein, suggesting calcium ions are released just prior to Hcen1 E105K’s denaturation temperature.
Furthermore, the isolation of a highly insoluble human Sfi1 (hsfi1) D10 construct for future conformational dynamic studies with the mutant hcen1protein formerly described was performed using a GST affinity column. Purification of this construct has allowed us to endeavor co-isolation of hsfi1-hcen complexes and higher order hsfi1 domains using analogous purification methods.
