Tuning protein mechanics through an ionic cluster graft from an extremophilic protein

Tych, Katarzyna M.; Batchelor, Matthew; Hoffmann, T. A.; Wilson, Michael C.; Paci, Emanuele; Brockwell, David J.; Dougan, Lorna

doi:10.1039/c5sm02938d

Cited by 11 publications

(44 citation statements)

References 78 publications

(108 reference statements)

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“…For that reason, mechanical properties of cold shock proteins have been extensively studied both experimentally and in simulations. 33,[52][53][54][55] Moreover, the unfolding of CspA under force and high temperature was previously investigated by our group using all-atom simulations, 33 provinding an interesting reference for the present work.…”

Section: Systemmentioning

confidence: 93%

Three Weaknesses for Three Perturbations: Comparing Protein Unfolding Under Shear, Force, and Thermal Stresses

Languin–Cattoën

Melchionna²,

Derreumaux

et al. 2018

J. Phys. Chem. B

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The perturbation of a protein conformation by a physiological fluid flow is crucial in various biological processes including blood clotting and bacterial adhesion to human tissues. Investigating such mechanisms by computer simulations is thus of great interest but it requires to develop ad hoc strategies to mimic the complex hydrodynamic interactions acting on the protein from the surrounding flow. In this study, we apply the Lattice Boltzmann Molecular Dynamics (LBMD) technique built on the implicit solvent coarse-grained model for protein OPEP and a mesoscopic representation of the fluid solvent, to simulate the unfolding of a small globular cold shock protein in shear flow, and to compare it to the unfolding mechanisms caused either by mechanical or thermal perturbations. We show that each perturbation probes a specific weakness of the protein, and causes the disruption of the native fold along different unfolding pathways. Notably, the shear flow and the thermal unfolding exhibit very similar pathways, while because of the directionality of the perturbation, the unfolding under force is quite different. For force and thermal disruption of the native state, the coarse-grained simulations are compared to all-atom simulations in explicit solvent, showing an excellent agreement in the explored unfolding mechanisms. These findings encourages the use of LBMD based on the OPEP model to investigate how a flow can affect the function of larger proteins, e.g. in catch-bond systems.

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Section: Systemmentioning

confidence: 93%

Three Weaknesses for Three Perturbations: Comparing Protein Unfolding Under Shear, Force, and Thermal Stresses

Languin–Cattoën

Melchionna²,

Derreumaux

et al. 2018

J. Phys. Chem. B

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“…Polyproteins were constructed using a method which makes use of Gibson assembly cloning and purified using a method described previously, including an additional stage to remove any bound nucleic acid . Three (His) 6 -tagged chimeric polyprotein constructs, each containing four domains of I27 interdigitated with three domains of a CSP, were produced: (i) a polyprotein containing the wild-type CSP from the hyperthermophilic organism T.…”

Section: Methodsmentioning

confidence: 99%

“…Fluorescence spectra were measured in a 1 cm path length quartz cuvette using an excitation wavelength of 280 nm and emission range of 320–380 nm with a 1 nm step size. Unfolding transitions were followed by a change in the barycentric median (BCM) as described previously. , The BCM “center of mass” of each spectrum between 320 and 380 nm was calculated, where I (λ) is the fluorescence value at a respective wavelength. The BCM value for each spectrum was plotted against temperature ( T ) or denaturant concentration ([GdnHCl]), and the unfolding transition was followed by an increase in BCM due to a shift to a higher wavelength of the unfolded peak.…”

Section: Methodsmentioning

confidence: 99%

“…Homologous proteins from hyperthermophilic and mesophilic organisms therefore offer important model systems with which to investigate the importance of specific noncovalent interactions for protein stability. − We recently took this approach to examine the role of salt bridges in the stability of a homologous pair, the cold shock protein B ( Tm CSP) from the hyperthermophilic organism T. maritima and the cold shock protein ( Bs CSP) from the mesophilic organism Bacillus subtilis.…”

Section: Introductionmentioning

confidence: 99%

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Differential Effects of Hydrophobic Core Packing Residues for Thermodynamic and Mechanical Stability of a Hyperthermophilic Protein

et al. 2016

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Article:Tych, KM, Batchelor, M orcid.org/0000-0001-6338-5698, Hoffmann, T et al. KEYWORDS AFM, extremophile, mechanical stability, cold shock proteins ABSTRACT Proteins from organisms which have adapted to environmental extremes provide attractive systems to explore and determine the origins of protein stability. Improved hydrophobic core packing and decreased loop-length flexibility can increase the thermodynamic stability of proteins from hyperthermophilic organisms. However, their impact on hyperthermophilic protein mechanical stability is not known. Here, we use protein engineering, biophysical characterization, single molecule force spectroscopy (SMFS) and molecular dynamics (MD) simulations to measure the effect of altering hydrophobic core packing on the stability of the cold shock protein TmCSP from the hyperthermophilic bacterium Thermotoga maritima. We make two variants of TmCSP in which a mutation is made to reduce the size of aliphatic groups from buried hydrophobic side chains. In the first, a mutation is introduced in a long loop (TmCSP L40A); in the other, the mutation is introduced on the C-terminal -strand (TmCSP V62A). We use MD simulations to confirm that the mutant TmCSP L40A shows the most significant increase in loop flexibility, and mutant TmCSP V62A shows greater disruption to the core packing. We measure the thermodynamic stability (∆GD-N) of the mutated proteins and show there is a more significant reduction for TmCSP L40A (∆∆G = 63%) than TmCSP V62A (∆∆G = 47%) as might be expected, based on the relative reduction in the size of the side chain. By contrast SMFS measures the mechanical stability (∆G*) and shows a greater reduction for TmCSP V62A (∆∆G* = 8.4%) than TmCSP L40A (∆∆G* = 2.5%). While the impact on mechanical stability is subtle, the results demonstrate the power of tuning non-covalent interactions to modulate both the thermodynamic and mechanical stability of a protein. Such understanding and control provides the opportunity to design proteins with optimized thermodynamic and mechanical properties. INTRODUCTIONProteins from organisms adapted to high-temperatures have evolved to retain their native, folded structure and function in the challenging environments in which the organisms grow.

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“…In a recent example, we demonstrated that a protein from a hyperthermophilic organism can be used to determine the role of salt bridge interactions in determining protein stability [238]. SMFS experiments showed that at ambient temperatures TmCsp is mechanically stronger yet, counter-intuitively, its native state can withstand greater deformation before unfolding (i.e.…”

Section: Lessons From Extreomophiles: Smfs Studies Of Extreme-loving mentioning

confidence: 99%

The physics of pulling polyproteins: a review of single molecule force spectroscopy using the AFM to study protein unfolding

2016

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One of the most exciting developments in the field of biological physics in recent years is the ability to manipulate single molecules and probe their properties and function. Since its emergence over two decades ago, single molecule force spectroscopy has become a powerful tool to explore the response of biological molecules, including proteins, DNA, RNA and their complexes, to the application of an applied force. The force versus extension response of molecules can provide valuable insight into its mechanical stability, as well as details of the underlying energy landscape. In this review we will introduce the technique of single molecule force spectroscopy using the atomic force microscope (AFM), with particular focus on its application to study proteins. We will review the models which have been developed and employed to extract information from single molecule force spectroscopy experiments. Finally, we will end with a discussion of future directions in this field.

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Tuning protein mechanics through an ionic cluster graft from an extremophilic protein

Cited by 11 publications

References 78 publications

Three Weaknesses for Three Perturbations: Comparing Protein Unfolding Under Shear, Force, and Thermal Stresses

Three Weaknesses for Three Perturbations: Comparing Protein Unfolding Under Shear, Force, and Thermal Stresses

Differential Effects of Hydrophobic Core Packing Residues for Thermodynamic and Mechanical Stability of a Hyperthermophilic Protein

The physics of pulling polyproteins: a review of single molecule force spectroscopy using the AFM to study protein unfolding

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