The expression and conformation of bacterial proteins and peptides can be monitored in situ by Fourier transform infrared spectroscopy (FTIR), provided that the concentration of the protein of interest is sufficient. Here, we describe a simple protocol to analyze the conformation adopted by a specific amyloid protein in Escherichia coli cells, the pleiotropic regulator Hfq.E. coli cells expressing Hfq under an inducible promoter are analyzed. The change in protein conformation is analyzed by comparing the different populations versus controls (i.e., Δhfq cells, totally devoid of the Hfq protein) by difference spectroscopy, second derivation, curve-fitting, and principal component analysis. All the analyses were performed in the free, open-source software Quasar. We describe the detailed protocol for analyzing the data in Quasar. We show that the specific absorption of the β-amyloid conformation can be easily detected in the WT-Hfq, with bands at 1624 cm-1 and 1693 cm-1 indicating the presence of both parallel and antiparallel β-sheets. Furthermore, we show that FTIR spectroscopy is sensitive enough to probe the conformation of an amyloid protein backbone in vivo and to analyze its conformation in situ, directly in bacterial cells, without the need for protein purification.
Keywords: Bacterial amyloid; Hfq; In vivo analysis; Vibrational spectroscopy.
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