The Application of Crosslinkers in Monitoring the Dynamics of Protein Interactions
Doepker, Miranda J.
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Due to the importance and complexities of protein interactions in cellular function, proteomic research has pushed to develop new methods to efficiently mapping out interactions and studying their dynamics. Chemical crosslinking is one method of analysis, when used in conjunction with mass spectrometry provides researchers insight in to the dynamic interactions that take place among proteins in vivo. Several challenges however are still associated with the use of this method. Fragmentation of the crosslinker specifically during spectrometry causes ambiguity when determining interaction sites. To address this problem, this lab previously synthesized a new crosslinker, DC4, which successfully fragments in identifiable patterns allowing for better determination of the crosslinking sites. In this study further evaluation of DC4 was conducted to determine the reaction properties associated with the addition of a diamine group to the crosslinker. Upon investigation it was found that the amine addition did not reduce the amount of nonspecific interactions that occurred in solution however it did facilitate the crosslinking of ADH, which was not amiable to crosslinking by the commercialized crosslinker BS3. Furthermore, we used this crosslinker to study the affects of environmental conditions, specifically protein crowding and salt concentration, on protein complex formation. It was determined that crowding and salt concentration did not directly affect complex formation but DC4 used at higher concentrations for shorter periods of time did result in reduced non specific interactions during the mixed protein trials. The mixed protein trial results further offered indication that crosslinkers may be a useful tool in the study of protein reaction rates.