Conformational Fluctuations As An Intrinsic Mechanism Of Action: The Lipase-Specific Foldase Of Pseudomonas Aeruginosa Studied By Hybrid Fluorescence Spectroscopy And Md Simulations

The description of protein function requires structural information, not only from static structural models but also of flexibility and dynamics. An investigation of highly flexible proteins or IDPs requires techniques sensitive to a variety of time- and distance-scales. Fluorescence (FRET, FCS) is known to provide good temporal and spatial coverage but it is a label-based technique and lacks atomistic-details. All-atom MD, on the other hand, is challenged by simulation of slow processes and accurate description of all interactions. The lipase-specific foldase (Lif) from Pseudomonas aeruginosa is a highly flexible protein with residual secondary and tertiary structure. It is mandatory to produce lipase A (LipA) in an enzymatically active conformation. Using a combination of fluorescence techniques (single molecule FRET, fluorescence lifetime analysis and fFCS) and all-atom MD we propose a description of structure and dynamics of Lif in relation to LipA. In the Lif:LipA complex, Lif forms flexible α-helical scaffold embracing LipA in headphone-like structure. In the unbound form, Lif does not stay in the hollow “headphone” conformation but rather exhibits large-scale conformational dynamics, where its α-helical structure undergoes reversible collapses and extensions as well as unfolding, on the sub-microsecond to sub-millisecond timescale. This process allows Lif to bind LipA despite the fickleness of structure. We show how the multitude of states in fast exchange provides a basis for binding. In addition we show how this hybrid approach benefits FRET (insight into the molecular structure) and MD (sampling).