Thursday, November 21, 2024
11/21/2024
SUMMARY Multiple chromodomain-helicase-DNA-binding (CHD) proteins have been implicated in rare diseases. While CHD proteins are functionally known to be DNA helicases and chromatin remodeling factors that can regulate transcription, insight into their structure and how it influences function remains sparse. Even AlphaFold2 models for these proteins have large regions of low confidence (over 60% of residues in CHD8 have pLDDT scores less than 70). Our long-term goal is to understand the molecular features of these proteins and how they impact Chiari Malformation such that better diagnostic and treatment approaches can be realized. The primary goal of this proposal is to produce full-length wild type and disease related variants of three CHD proteins on NIH’s list of understudied proteins related to rare disease and begin their biochemical and structural characterization. Our hypothesis is that in vitro cell-free expression can overcome the primary bottleneck to producing these proteins to begin deeper molecular characterization studies. The rationale underlying the proposed work is that cell-free expression allows small scale, versatile and rapid optimization of conditions to increase the chances of synthesizing a full-length protein that has similar solubility, biochemical and other characteristics as the native protein. Two specific aims will be pursued to complete this work: 1) Employ cell-free expression to synthesize CHD2, CHD7, and CHD8 as full-length wild-type proteins along with 2 disease related variants of each. 2) Begin deep profiling of synthesized proteins by evaluating protein-protein binding and DNA binding and initiate structural characterization. The success of the project will hinge on recent innovations in our cell-free expression pipeline including the ability to perform both functional binding and structural studies from cell-free expression reactions of only 25-microliter volume and without purification. The proposed research is significant since it will reveal new molecular details about 3 understudied proteins linked to multiple rare diseases. It will also lay the foundation for a workflow extending our cell-free expression pipeline from bioenergy research and into the biomedical field to permit its application toward other understudied proteins in the future. The expected outcome of this work is the synthesis of wild type and mutant variants of CHD2, CHD7 and CHD8 along with initial biochemical and structural characterization. Follow-on projects are expected to provide more in-depth molecular phenotyping. The results will immediately impact the study of Chiari Malformation and advance the understanding of the CHD family of proteins.
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