Kinetics of breaking a salt-bridge critical in protein unfolding

2004 ◽  
Vol 385 (5-6) ◽  
pp. 337-340 ◽  
Author(s):  
Andreea D. Gruia ◽  
Stefan Fischer ◽  
Jeremy C. Smith
Keyword(s):  
Protein Unfolding ◽  
Salt Bridge ◽  
Kinetics Of

scholarly journals A tryptophan synchronous and normal fluorescence study on bacteria inactivation mechanism

2019 ◽  
Vol 116 (38) ◽  
pp. 18822-18826 ◽  
Author(s):  
Runze Li ◽  
Dinesh Dhankhar ◽  
Jie Chen ◽  
Thomas C. Cesario ◽  
Peter M. Rentzepis
Keyword(s):  
Fluorescence Intensity ◽  
Protein Unfolding ◽  
Uv Light ◽  
Quantum Yields ◽  
Continuous Irradiation ◽  
Minute Period ◽  
Bacteria Inactivation ◽  
Tryptophan Residues ◽  
Inactivation Mechanism ◽  
Kinetics Of

The UV photodissociation kinetics of tryptophan amino acid, Trp, attached to the membrane of bacteria, Escherichia coli and Bacillus subtilis, have been studied by means of normal and synchronous fluorescence. Our experimental data suggest that the fluorescence intensity of Trp increases during the first minute of irradiation with 250 nm to ∼ 280 nm, 7 mW/cm2 UV light, and subsequently decreases with continuous irradiation. During this short, less than a minute, period of time, 70% of the 107 cell per milliliter bacteria are inactivated. This increase in fluorescence intensity is not observed when tryptophan is in the free state, namely, not attached to a protein, but dissolved in water or saline solution. This increase in fluorescence is attributed to the additional fluorescence of tryptophan molecules formed by protein unfolding, the breakage of the bond that attaches Trp to the bacterial protein membrane, or possibly caused by the irradiation of 2 types of tryptophan residues that photolyze with different quantum yields.

scholarly journals The Trajectory Taken by Dimeric Cu/Zn Superoxide Dismutase Through the Protein Unfolding and Dissociation Landscape Is Modulated by Salt-Bridge Formation

2019 ◽  
Author(s):  
Luke McAlary ◽  
Julian Harrison ◽  
J. Andrew Aquilina ◽  
Steven Fitzgerald ◽  
Celine Kelso ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Free Energy ◽  
Gas Phase ◽  
Protein Unfolding ◽  
Energy Landscape ◽  
Salt Bridge ◽  
Native Mass Spectrometry ◽  
Free Energy Landscape ◽  
Related Point ◽  
Macromolecular Protein

<p>Native mass spectrometry (MS) is a powerful means for studying macromolecular protein assemblies, including accessing activated states. However, much remains to be understood about what governs which regions of the protein (un)folding funnel are explored by activation of protein ions in vacuum. Here we examine the trajectory that dimeric Cu/Zn superoxide dismutase (SOD1) dimers take over the unfolding and dissociation free energy landscape in vacuum. We examined wild-type SOD1 and six disease-related point-mutants by using tandem MS and ion-mobility MS (MS/MS-IMMS) coupled with increasing collisional activation potentials. For six of the seven SOD1 variants, increasing activation promoted dimers to transition through two unfolding events to access three gas-phase conformers before dissociating symmetrically into monomers with (as near as possible) equal charges. The exception was G37R, which proceeded only through the first unfolding transition, and displayed a much higher abundance of asymmetric products. We localise this effect to the formation of a new salt-bridge in the first activated conformation. To examine the data quantitatively, we generated a model of SOD1 gas phase unfolding and dissociation, and applied Arrhenius-type analysis to estimate the barriers on the corresponding free energy landscape. This reveals an increase in the barrier height to unfolding in G37R to be >5 kJ/mol<sup>-1</sup> higher than for the other variants, consistent with expectations for the strength of a salt-bridge. Our work demonstrates the importance of bond formation during the unfolding of proteins in vacuum, and provides a framework for comparing quantitatively the free energy landscape they explore upon activation.</p>

scholarly journals The Trajectory Taken by Dimeric Cu/Zn Superoxide Dismutase Through the Protein Unfolding and Dissociation Landscape Is Modulated by Salt-Bridge Formation

2019 ◽  
Author(s):  
Luke McAlary ◽  
Julian Harrison ◽  
J. Andrew Aquilina ◽  
Steven Fitzgerald ◽  
Celine Kelso ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Free Energy ◽  
Gas Phase ◽  
Protein Unfolding ◽  
Energy Landscape ◽  
Salt Bridge ◽  
Native Mass Spectrometry ◽  
Free Energy Landscape ◽  
Related Point ◽  
Macromolecular Protein

<p>Native mass spectrometry (MS) is a powerful means for studying macromolecular protein assemblies, including accessing activated states. However, much remains to be understood about what governs which regions of the protein (un)folding funnel are explored by activation of protein ions in vacuum. Here we examine the trajectory that dimeric Cu/Zn superoxide dismutase (SOD1) dimers take over the unfolding and dissociation free energy landscape in vacuum. We examined wild-type SOD1 and six disease-related point-mutants by using tandem MS and ion-mobility MS (MS/MS-IMMS) coupled with increasing collisional activation potentials. For six of the seven SOD1 variants, increasing activation promoted dimers to transition through two unfolding events to access three gas-phase conformers before dissociating symmetrically into monomers with (as near as possible) equal charges. The exception was G37R, which proceeded only through the first unfolding transition, and displayed a much higher abundance of asymmetric products. We localise this effect to the formation of a new salt-bridge in the first activated conformation. To examine the data quantitatively, we generated a model of SOD1 gas phase unfolding and dissociation, and applied Arrhenius-type analysis to estimate the barriers on the corresponding free energy landscape. This reveals an increase in the barrier height to unfolding in G37R to be >5 kJ/mol<sup>-1</sup> higher than for the other variants, consistent with expectations for the strength of a salt-bridge. Our work demonstrates the importance of bond formation during the unfolding of proteins in vacuum, and provides a framework for comparing quantitatively the free energy landscape they explore upon activation.</p>

scholarly journals Assaying the kinetics of protein denaturation catalyzed by AAA+ unfolding machines and proteases

2015 ◽  
Vol 112 (17) ◽  
pp. 5377-5382 ◽  
Author(s):  
Vladimir Baytshtok ◽  
Tania A. Baker ◽  
Robert T. Sauer
Keyword(s):  
Protein Unfolding ◽  
Protein Denaturation ◽  
Molecular Machines ◽  
The Self ◽  
The Other ◽  
Robust Methods ◽  
Specific Proteins ◽  
Dimeric Subunit ◽  
Kinetics Of

ATP-dependent molecular machines of the AAA+ superfamily unfold or remodel proteins in all cells. For example, AAA+ ClpX and ClpA hexamers collaborate with the self-compartmentalized ClpP peptidase to unfold and degrade specific proteins in bacteria and some eukaryotic organelles. Although degradation assays are straightforward, robust methods to assay the kinetics of enzyme-catalyzed protein unfolding in the absence of proteolysis have been lacking. Here, we describe a FRET-based assay in which enzymatic unfolding converts a mixture of donor-labeled and acceptor-labeled homodimers into heterodimers. In this assay, ClpX is a more efficient protein-unfolding machine than ClpA both kinetically and in terms of ATP consumed. However, ClpP enhances the mechanical activities of ClpA substantially, and ClpAP degrades the dimeric substrate faster than ClpXP. When ClpXP or ClpAP engage the dimeric subunit, one subunit is actively unfolded and degraded, whereas the other subunit is passively unfolded by loss of its partner and released. This assay should be broadly applicable for studying the mechanisms of AAA+ proteases and remodeling chaperones.

Kinetics of protein unfolding at interfaces

2012 ◽  
Vol 24 (50) ◽  
pp. 503101 ◽  
Author(s):  
Yohko F Yano
Keyword(s):  
Protein Unfolding ◽  
Kinetics Of

KINETICS OF HYDROLYSIS OF THE CHROMOGENIC SUBSTRATE S2238 BY ALPHA-THROMBIN : INFLUENCE OF THE pH AND THE IONIC STRENGTH

1987 ◽  
Author(s):  
I M A Verhamme ◽  
G W K van Dedem
Keyword(s):  
Ionic Strength ◽  
Salt Bridge ◽  
Reaction Conditions ◽  
Ionic Strength Dependence ◽  
Strength Dependence ◽  
Activity Decrease ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Optimal Reaction ◽  
Ph Profiles

The knowledge of the pH and ionic strength dependence of kcat and Km for the hydrolysis of S2238 (HD-phe-pip-arg-pNA.2HC1) by alpha-thrombin is essential in determining optimal reaction conditions of residual enzyme in systems where also protease inhibitors and glycosaminoglycan catalysts play a role.We studied the kinetic behavior of S2238 in piperazine/glycylglycine/NaOH buffers from pH 6 to 11 and with a calculated ionic strength up to 0.7 M taking into account the pH-dependent concentration of the buffer species. The kinetic parameters of 60 Michaelis-Menten substrate functions were used for the setup of ionic strength and pH profiles. The kcat values are dependent upon the ionic strength, increasing steeply up to about 0.3 M and decreasing again at high ionic strength.The Km however,reflecting the affinity between enzyme and substrate,is nearly unaffected.The Km values at very alkaline pH are markedly elevated,indicating a conformational form which does not readily bind substrate.The pH profiles for kcat and kcat/Km are displaced towards the low pH side with increasing ionic strength.The ascending limb corresponds to the pK of the Asp-His charge relay system,decreasing with increasing ionic strength from 7.2 to 6.6 in the ES complex and from 6.8 to 6.6 in the free enzyme.Apparently substrate binding provokes a pK increase of the active His residue.The descending limb in the kcat profile could be described by a hypothetical pK varying from 11.5 to 10.7 but the activity decrease is probably due to enzyme inactivation.The alkaline limb of the kcat/Km profile is governed by a pK of 9.4 which is rather independent of the ionic strength and could be attributed to the B-chain terminal isoleucine ,forming a salt bridge with Asp 194 and stabilizing the active site conformation as proven for other serine proteases.Data analysis via a modified Debije function with appropriate estimates for the dielectric constant and the radius of the macro-ion can provide information on the charge density of the enzyme.

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