Functional Assessment of Protein Variants in Structured Domains by Fluorescence Cross-Correlation Spectroscopy

Abstract

With the expanding catalogue of novel disease-genes, there is an increasing need to establish the clinical significance of potential disease-causing variants. Based on the idea that pathogenic variants in structured protein domains disturb folding and association with macromolecular assemblies, we employed Fluorescence Correlation and Cross-Correlation Spectroscopy (FCS and FCCS) to assess in vivo protein complex formation. Since the molecular underpinning of BRCA-associated breast and ovarian cancers is well defined and data from a recent genome editing screening allowed us to compare the binding data with a reliable functional HRD test, we examined the binding of BRCA1 to BARD1 and RBBP8, respectively. The results demonstrate that FCCS, whether applied to full-length BRCA1 in live cells or to isolated domains in cellular lysates, reliably identified pathogenic BRCA1 RING or BRCT variants. We moreover demonstrate the feasibility of employing FCCS for analysis of HNPCC-related factor MSH2 and MEN1 factor Menin variants in combination with DNA mismatch repair factor MSH6 and transcription factor JUND, respectively. Because the procedure can be completed within a clinically relevant time frame, FCCS is an appealing complement to current clinical procedures for classifying variants. Given its generic nature and design, the approach can be applied to a variety of monogenic diseases.