Conditional Chaperone-Client Interactions Revealed by Genetically Encoded Photo-cross-linkers.

Gram-negative bacteria face harsh extracellular conditions, including acidic environments. The periplasmic space lacks ATP energy systems, making it difficult to maintain biological activity under stress. Enteric pathogens rely on conditionally disordered chaperones like HdeA and HdeB to survive acidic conditions. These chaperones lose their ordered structures in response to environmental stress, enabling promiscuous binding to client proteins. However, the client specificity of these chaperones remains poorly understood. Existing tools lack the ability to capture and compare dynamic interactions in living cells. Prior research has shown that genetically encoded photo-cross-linkers can capture protein interactions, but their use in profiling condition-dependent client pools is limited.

This work aimed to develop genetically encoded photo-cross-linkers to study disorder-mediated chaperone-client interactions in living cells. The goal was to systematically compare the client profiles of HdeA and HdeB and map their interaction interfaces. The researchers sought to address the lack of tools for profiling and comparing condition-dependent client pools from homologous chaperones. By using proteomic-based strategies, they aimed to reveal the mode of action and client specificity of these chaperones. The study also aimed to evaluate the effectiveness of photo-cross-linkers in capturing promiscuous binding partners and identifying interaction interfaces. The approach was motivated by the need to understand how conditionally disordered chaperones function in bacterial survival.

The researchers developed a panel of genetically encoded photo-cross-linkers to study chaperone-client interactions in living cells. These tools were used to capture and compare the client profiles of HdeA and HdeB. Proteomic-based strategies were employed to systematically analyze the client pools under varying conditions. The photo-cross-linkers enabled the identification of interaction interfaces between chaperones and their clients. The methods focused on profiling the condition-dependent client pools from homologous chaperones. The approach allowed for side-by-side comparisons of HdeA and HdeB interactions. The study combined genetic encoding with proteomic analysis to map binding interfaces. The methods were designed to capture dynamic and promiscuous interactions in physiological settings.

The study revealed the client specificity of HdeA and HdeB under acidic conditions. The photo-cross-linkers successfully captured promiscuous binding partners and identified interaction interfaces. The researchers found that HdeA and HdeB bind to a wide range of acid-denatured proteins. The proteomic analysis showed distinct client profiles for each chaperone. The results indicated that these chaperones lose their ordered structures in response to acid stress. The study demonstrated that the chaperones protect diverse proteins by forming transient interactions. The findings showed that HdeA and HdeB have overlapping but distinct client pools. The data suggest that these chaperones function through a disorder-mediated mechanism.

The study demonstrated that genetically encoded photo-cross-linkers can capture disorder-mediated chaperone-client interactions in living cells. The findings revealed the client specificity of HdeA and HdeB under acidic conditions. The researchers showed that these chaperones bind to a wide range of acid-denatured proteins. The study provided insights into the mode of action of conditionally disordered chaperones. The results suggest that HdeA and HdeB have overlapping but distinct client pools. The use of proteomic-based strategies allowed for the systematic comparison of chaperone interactions. The findings support the hypothesis that these chaperones function through a disorder-mediated mechanism. The study highlights the potential of photo-cross-linkers in profiling condition-dependent protein interactions.