Direct Observation of T4 Lysozyme Hinge-Bending Motion by Fluorescence Correlation Spectroscopy
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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.
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Fluorescence correlation analysis indicates that T4 lysozyme populates multiple intermediate states.
“Correlation analysis of fluorescence indicates that T4L populates multiple intermediate states.222”
From Protein Ensembles: How Does Nature Harness Thermodynamic Fluctuations for Life? The Diverse Functional Roles of Conformational Ensembles in the Cell (2016) · cites this paper as [222]
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- Authors
- Yirdaw, R. B., & Mchaourab, H. S.
- Affiliations
- Vanderbilt University (US)
- Corresponding author
- Mchaourab, Hassane S.
- Venue
- Biophysical Journal
- Publisher
- Elsevier BV
- Published
- 2012-10
- Volume
- 103
- Issue
- 7
- Pages
- 1525-1536
- ISSN
- 0006-3495 (ISSN-L)
- PMC
- PMC3471472
- OpenAlex
- W2010047075
- Cited by
- 29
- References
- 55
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 1 · 2023: 2 · 2022: 4 · 2021: 2 · 2020: 4 · 2018: 1 · 2017: 2 · 2016: 2 · 2015: 3 · 2014: 3 · 2013: 2 · 2012: 1
- FWCI
- 1.16
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Biophysical Journal (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Eye Institute (R29EY012018, R01EY012018, EY12018)
- National Institute of General Medical Sciences (T32GM008320)
- National Center for Research Resources (S10RR21208, S10RR021208)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
- Keywords
- bacteriophage T4 lysozyme, hinge-bending motion, conformational equilibrium, open and closed conformations, fluorescence fluctuation spectroscopy, relaxation time, substrate trapping, peptidoglycan hydrolysis, enzyme dynamics
- MeSH terms
- Kinetics, Movement, Muramidase, Spectrometry, Fluorescence, Bacteriophage T4, Protein Structure, Secondary, Protein Structure, Tertiary, Molecular Dynamics Simulation
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