scholarly journals Origin and Prediction of Highly Specific Bond Cleavage Sites in the Thermal Activation of Intact Protein Ions

2018 ◽  
Vol 25 (3) ◽  
pp. 823-834 ◽  
Author(s):  
Huixin Wang ◽  
Michael G. Leeming ◽  
Junming Ho ◽  
William A. Donald
Keyword(s):  
Thermal Activation ◽  
Bond Cleavage ◽  
Intact Protein ◽  
Cleavage Sites ◽  
Protein Ions

scholarly journals Tilting a ground-state reactivity landscape by vibrational strong coupling

Science ◽  
2019 ◽  
Vol 363 (6427) ◽  
pp. 615-619 ◽  
Author(s):  
A. Thomas ◽  
L. Lethuillier-Karl ◽  
K. Nagarajan ◽  
R. M. A. Vergauwe ◽  
J. George ◽  
...  
Keyword(s):  
Strong Coupling ◽  
Activation Barrier ◽  
Bond Cleavage ◽  
Optical Cavity ◽  
Vacuum Field ◽  
Activation Entropy ◽  
Vibrational Modes ◽  
Cleavage Sites ◽  
Chemical Methods ◽  
Site Selectivity

Many chemical methods have been developed to favor a particular product in transformations of compounds that have two or more reactive sites. We explored a different approach to site selectivity using vibrational strong coupling (VSC) between a reactant and the vacuum field of a microfluidic optical cavity. Specifically, we studied the reactivity of a compound bearing two possible silyl bond cleavage sites—Si–C and Si–O, respectively—as a function of VSC of three distinct vibrational modes in the dark. The results show that VSC can indeed tilt the reactivity landscape to favor one product over the other. Thermodynamic parameters reveal the presence of a large activation barrier and substantial changes to the activation entropy, confirming the modified chemical landscape under strong coupling.

scholarly journals Bovine Complex I Is a Complex of 45 Different Subunits

2006 ◽  
Vol 281 (43) ◽  
pp. 32724-32727 ◽  
Author(s):  
Joe Carroll ◽  
Ian M. Fearnley ◽  
J. Mark Skehel ◽  
Richard J. Shannon ◽  
Judy Hirst ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Mass ◽  
Electrospray Ionization ◽  
Complex I ◽  
Bond Cleavage ◽  
Peptide Bond ◽  
Intact Protein ◽  
Ionization Mass ◽  
Peptide Bond Cleavage ◽  
Membrane Bound

Mammalian mitochondrial complex I is a multisubunit membrane-bound assembly with a molecular mass approaching 1 MDa. By comprehensive analyses of the bovine complex and its constituent subcomplexes, 45 different subunits have been characterized previously. The presence of a 46th subunit was suspected from the consistent detection of a molecular mass of 10,566 by electrospray ionization mass spectrometry of subunits fractionated by reverse-phase high pressure liquid chromatography. The component was found associated with both the intact complex and subcomplex Iβ, which represents most of the membrane arm of the complex, and it could not be resolved chromatographically from subunit SGDH (the subunit of bovine complex I with the N-terminal sequence Ser-Gly-Asp-His). It has now been characterized by tandem mass spectrometry of intact protein ions and shown to be a C-terminal fragment of subunit SGDH arising from a specific peptide bond cleavage between Ile-55 and Pro-56 during the electrospray ionization process. Thus, the subunit composition of bovine complex I has been established. It is a complex of 45 different proteins plus non-covalently bound FMN and eight iron-sulfur clusters.

scholarly journals Tilting a Ground State Reactivity Landscape by Vibrational Strong Coupling

2018 ◽  
Author(s):  
Anoop Thomas ◽  
Lucas Lethuillier-Karl ◽  
Kalaivanan Nagarajan ◽  
Robrecht M. A. Vergauwe ◽  
Jino George ◽  
...  
Keyword(s):  
Strong Coupling ◽  
Activation Barrier ◽  
Chemical Reactivity ◽  
Bond Cleavage ◽  
Optical Cavity ◽  
Vacuum Field ◽  
Activation Entropy ◽  
Cleavage Sites ◽  
Site Selectivity ◽  
First Time

Site-selectivity is fundamental for steering chemical reactivity towards a given product and various efficient chemical methods have been developed for this purpose. Here we explore a very different approach by using vibrational strong coupling (VSC) between a reactant and the vacuum field of a microfluidic optical cavity. For this purpose, the reactivity of a compound bearing two possible silyl bond cleavage sites, at Si-C and Si-O, was studied as a function of VSC of its various vibrational modes in the dark. The results show that VSC can indeed tilt the reactivity landscape to favor one product over the other. Thermodynamic parameters reveal the presence of a large activation barrier and significant changes to the activation entropy, confirming the modified chemical landscape under strong coupling. This study shows for the first time that VSC can impart site-selectivity for chemical reactions without the need for chemical intervention.

The Substrate -Membrane Interaction Constrains Bond Presentation and Cleavage in the Action of Prothrombinase on Prothrombin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1712-1712
Author(s):  
Steven J. Orcutt ◽  
Sriram Krishnaswamy
Keyword(s):  
Active Site ◽  
Bond Cleavage ◽  
Membrane Binding ◽  
Product Formation ◽  
Cleavage Sites ◽  
Membrane Interaction ◽  
Active Product ◽  
Fold Reduction ◽  
The Individual ◽  
Prothrombin Activation

Abstract Thrombin formation is catalyzed by human prothrombinase almost exclusively through sequential cleavage at Arg320 followed by cleavage at Arg271 in prothrombin. Activation site mutants in which the individual sites have been rendered uncleavable indicate that Arg271 in intact prothrombin is hydrolyzed ~26-fold more slowly than Arg320. This finding provides an adequate kinetic explanation for the observed pathway of prothrombin activation by prothrombinase via the formation of meizothrombin. However, deviations from this behavior are implied by findings in the absence of membranes or with prothrombin species that are defective in membrane binding. We have investigated the contribution of the substrate-membrane interaction to the individual cleavage reactions by comparing the action of prothrombinase on a series of recombinant prothrombin derivatives expressed in the presence or absence of reduced vitamin K to yield either fully-carboxylated (Gla) or un-carboxylated (desGla) protein. The variants included wild type prothrombin (IIWT) containing both cleavage sites, IIQ271 with a single cleavable site at Arg320 and IIQ320 with a single cleavable site at Arg271. Quantitative analysis of 4-carboxyglutamic acid content established the intended extent of carboxylation for all Gla and desGla isoforms. Analysis of prothrombin activation by prothrombinase indicated a ~10 fold reduction in the rate of active product formation with desGla-IIWT compared to Gla-IIWT. Surprisingly, analysis by SDS-PAGE and quantitative densitometry revealed only a modest decrease (~ 2.5-fold) in the rate of consumption of desGla-IIWT. However, marked differences were evident in the intermediates produced. Bond cleavage of desGla-IIWT proceeded with a major contribution from cleavage at Arg271 followed by Arg320, in an order opposite to that observed with Gla-IIWT. Thus, the reduced rate of active product formation with desGla-IIWT reflects, in large part, the formation of a zymogen intermediate as a result of an altered order in which bonds are cleaved by prothrombinase. Comparison of the rates of cleavage of individual bonds in Gla and desGla versions of IIQ271 and IIQ320 revealed the kinetic basis for this change. Elimination of membrane binding led to a ~5 fold reduction in the rate of cleavage at Arg320 but surprisingly yielded a ~10-fold enhancement in cleavage at Arg271. Lack of Gla is not uniformly deleterious to all reactions of prothrombin activation. Compensating rate effects on the individual reactions imply a change in the restricted presentation of cleavage sites to the active site of Xa within prothrombinase upon the loss of the substrate-membrane interaction. Thus, substrate-membrane interactions appear to work in concert with exosite binding to modulate bond cleavage order by maximizing accessibility of the Arg320 bond to the active site while restricting that of Arg271. Loss of the membrane anchoring component leads to increased accessibility of the Arg271 site and a concomitant decrease in the rate of cleavage at Arg320. These observations have major implications for prothrombinase function on cell surfaces with limited binding sites for prothrombin, as well as the impact of warfarin therapy on prothrombinase function in vivo.

scholarly journals Proteolytic processing of the serine protease matriptase-2: identification of the cleavage sites required for its autocatalytic release from the cell surface

2010 ◽  
Vol 430 (1) ◽  
pp. 87-95 ◽  
Author(s):  
Marit Stirnberg ◽  
Eva Maurer ◽  
Angelika Horstmeyer ◽  
Sonja Kolp ◽  
Stefan Frank ◽  
...  
Keyword(s):  
Cell Surface ◽  
Disulfide Bonds ◽  
Serine Proteases ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Bond Cleavage ◽  
Proteolytic Processing ◽  
Cleavage Sites ◽  
Single Chain

Matriptase-2 is a member of the TTSPs (type II transmembrane serine proteases), an emerging class of cell surface proteases involved in tissue homoeostasis and several human disorders. Matriptase-2 exhibits a domain organization similar to other TTSPs, with a cytoplasmic N-terminus, a transmembrane domain and an extracellular C-terminus containing the non-catalytic stem region and the protease domain. To gain further insight into the biochemical functions of matriptase-2, we characterized the subcellular localization of the monomeric and multimeric form and identified cell surface shedding as a defining point in its proteolytic processing. Using HEK (human embryonic kidney)-293 cells, stably transfected with cDNA encoding human matriptase-2, we demonstrate a cell membrane localization for the inactive single-chain zymogen. Membrane-associated matriptase-2 is highly N-glycosylated and occurs in monomeric, as well as multimeric, forms covalently linked by disulfide bonds. Furthermore, matriptase-2 undergoes shedding into the conditioned medium as an activated two-chain form containing the catalytic domain, which is cleaved at the canonical activation motif, but is linked to a released portion of the stem region via a conserved disulfide bond. Cleavage sites were identified by MS, sequencing and mutational analysis. Interestingly, cell surface shedding and activation of a matriptase-2 variant bearing a mutation at the active-site serine residue is dependent on the catalytic activity of co-expressed or co-incubated wild-type matriptase-2, indicating a transactivation and trans-shedding mechanism.

scholarly journals The Autoproteolysis of Lactococcus lactis Lactocepin III Affects Its Specificity towards β-Casein

2000 ◽  
Vol 66 (12) ◽  
pp. 5134-5140 ◽  
Author(s):  
Benedicte Flambard ◽  
Vincent Juillard
Keyword(s):  
Liquid Chromatography ◽  
Lactococcus Lactis ◽  
High Performance ◽  
Bond Cleavage ◽  
Peptide Bond ◽  
Cleavage Sites ◽  
Peptide Bond Cleavage ◽  
Chromatography Analysis ◽  
On Line ◽  
Line Coupling

ABSTRACT The effect of autoproteolysis of Lactococcus lactislactocepin III on its specificity towards β-casein was investigated. β-Casein degradation was performed by using either an autolysin-defective derivative of L. lactis MG1363 carrying the proteinase genes of L. lactis SK11, which was unable to transport oligopeptides, or autoproteolyzed enzyme purified fromL. lactis SK11. Comparison of the peptide pools by high-performance liquid chromatography analysis revealed significant differences. To analyze these differences in more detail, the peptides released by the cell-anchored proteinase were identified by on-line coupling of liquid chromatography to mass spectrometry. More than 100 oligopeptides were released from β-casein by the cell-anchored proteinase. Analysis of the cleavage sites indicated that the specificity of peptide bond cleavage by the cell-anchored proteinase differed significantly from that of the autoproteolyzed enzyme.

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