Rodríguez-Nassif, Aslin Marie

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    Molecular biophysical characterization of a novel centrin-krr1 complex
    (2018) Rodríguez-Nassif, Aslin Marie; Pastrana-Ríos, Belinda; College of Arts and Sciences - Sciences; Meléndez, Enrique; Ríos, Jorge; Vera, Marisol; Department of Chemistry; Cruzado, Ivette
    The nucleus is the natural compartment for DNA in eukaryotes. This compartment is also the setting for genetic mutations, nucleotide excision repair (NER), and ribosome biogenesis. The study of protein-protein interactions (PPIs) and protein-DNA interactions is the key to understanding these processes in the cell. Abnormalities in ribosome biogenesis cause specific clinical syndromes and recently have been associated with tumorigenesis. One example involves the KH domain of Krr1, which has been identified as a single-stranded DNA (ssDNA) recognition motif. Centrin, an essential calcium binding protein, has been found to regulate NER within the nucleus. Krr1, a novel centrin target, is required for ribosome biogenesis. The structure of Homo sapiens Krr1 (HsKrr1) has not been determined to date. We describe the spectroscopic characterization of the GXXG loop peptide (GXXGlp), which is present in KH domain-containing proteins. The sequence of HsKrr1’s GXXGlp is evolutionarily conserved and has been associated with ssDNA interaction and ribosome biogenesis. 2D IR correlation spectroscopy was used to determine the stability of GXXGlp, as a synthetic peptide, and the optimal formulation conditions for the use of the peptide. We observed differences in the molecular behavior of GXXGlp in the presence and absence of trifluoroacetate at various peptide concentrations. 2D IR correlation spectroscopy was also used for the elucidation of the unfolding process, the mechanism and extent of peptide aggregation, and the effect of TFA on the stability of the peptide. This spectroscopic method can be applied to the characterization of any synthetic peptide. Furthermore, our findings indicate a GXXGlp-ssDNA interaction. We used the same technique to assess the stability of the complex via thermal perturbation. Preliminary studies identified a Hscentrin2-GXXGlp interaction using NMR, indicating that the interaction occurs in the C-terminal domain of Hscentrin, the Ca2+-dependent domain. New signals in the 2D HSQC centrin-GXXGlp titration experiments suggest that centrin adopts a new conformation upon binding to GXXGlp, exposing amino acids located in the EF hands of the C-terminal domain. Moreover, these studies identified a novel interaction between HsKrr1 and Hscentrin2’s C-terminal domain.