Direct Observation of T4 Lysozyme Hinge-Bending Motion by Fluorescence Correlation Spectroscopy

Abstract

Bacteriophage T4 Lysozyme (T4L) catalyzes the hydrolysis of the peptidoglycan layer of the bacterial cell wall late in the infection cycle. It has long been postulated that equilibrium dynamics enable substrate access to the active site located at the interface between the N- and C-terminal domains. Crystal structures of WT-T4L and point mutants captured a range of conformations that differ by the hinge-bending angle between the two domains. Evidence of equilibrium between open and closed conformations in solution was gleaned from distance measurements between the two domains but the nature of the equilibrium and the timescale of the underlying motion have not been investigated. Here, we used fluorescence fluctuation spectroscopy to directly detect T4L equilibrium conformational fluctuations in solution. For this purpose, Tetramethylrhodamine probes were introduced at pairs of cysteines in regions of the molecule that undergo relative displacement upon transition from open to closed conformations. Correlation analysis of Tetramethylrhodamine intensity fluctuations reveals hinge-bending motion that changes the relative distance and orientation of the N- and C-terminal domains with ≅ 15 μs relaxation time. That this motion involves interconversion between open and closed conformations was further confirmed by the dampening of its amplitude upon covalent substrate trapping. In contrast to the prevalent two-state model of T4L equilibrium, molecular brightness and number of particles obtained from cumulant analysis suggest that T4L populates multiple intermediate states, consistent with the wide range of hinge-bending angles trapped in the crystal structure of T4L mutants.

Observing Lysozyme’s Closing and Opening Motions by High-Resolution Single-Molecule Enzymology

“…14 However, the Δ I ( t ) signals corresponding to N >1 never contained two or more distinct levels. The long-lived pause in lysozyme’s motion was always associated with a single, intermediate configuration, even though statistical analysis indicated multiple consecutive processes.…”

“…FCS suggests that much larger data sets, such as those obtained by monitoring many molecules, might be necessary to reveal weak correlations amongst rare events. 14 …”

Distance Mapping in Proteins Using Fluorescence Spectroscopy: Tyrosine, like Tryptophan, Quenches Bimane Fluorescence in a Distance-Dependent Manner

“… 33 The latter distances were determined using a T4L active site mutant T26E, which covalently binds and traps the substrate peptidoglycan. 33 This active site mutant has been used in numerous previous studies focused on defining this hinge bending; 34 , 39 thus, here we tested if this hinge bending could be detected using TrIQ or TyrIQ, which would result in a decreased level of quenching upon substrate binding.…”

Structural Transformation of Bovine Serum Albumin Induced by Dimethyl Sulfoxide and Probed by Fluorescence Correlation Spectroscopy and Additional Methods

“…[26] Analysis of the correlation function using an appropriate model provides information on the processes that contribute to the fluctuations. [38,39] In the present work, we have exploited the fluorescence self-quenching process in fluorescein isothiocyanate (FITC)-labeled BSA to study the conformational dynamics of BSA. [33][34][35][36][37] Most of the FCS experiments performed earlier utilized electron/energy transfer between a labeled fluorophore and a part of the protein (tryptophan or heme group) to study the conformational dynamics of proteins.…”

“…There are few studies in which fluorescence self-quenching has been used for studying the conformational dynamics of proteins by using the FCS technique. [38,39] In the present work, we have exploited the fluorescence self-quenching process in fluorescein isothiocyanate (FITC)-labeled BSA to study the conformational dynamics of BSA. Additional steady-state measurements, such as the intrinsic fluorescence of BSA, fluorescence of 1-anilinonaphthalene-8-sulfonic acid (ANS), and the near-UV circular dichroism (CD) spectrum of BSA, validate the findings of the FCS measurements and provide additional information.…”