Structure and Behavior of Human α-Thrombin upon Ligand Recognition: Thermodynamic and Molecular Dynamics Studies

Silva, Vivian de Almeida; Cargnelutti, Maria Thereza; Giesel, Guilherme Menegon; Palmieri, L.C.; Monteiro, Robson Q.; Verli, Hugo; Lima, Luis Maurício Trambaioli da Rocha e

doi:10.1371/journal.pone.0024735

Cited by 8 publications

(6 citation statements)

References 58 publications

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“…56,57 Apart from these typical protein peaks, the peak at 926 cm −1 due to N−C α −C stretching of the α-helix and the peak at 1379 cm −1 due to tryptophan were also present in the SERS spectrum. 57 Human alpha thrombin is an alpharich protein, 58 the range of wavenumbers of Raman peaks of an alpha-rich protein are given in Table S4. The obtained peaks were matching with the literature.…”

mentioning

confidence: 99%

Broadband SERS Enhancement by DNA Origami Assembled Bimetallic Nanoantennas with Label-Free Single Protein Sensing

Tanwar¹,

Kaur²,

Kaur³

et al. 2021

J. Phys. Chem. Lett.

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Engineering hotspots in surface-enhanced Raman spectroscopy (SERS) through precisely controlled assembly of plasmonic nanostructures capable of expanding intense field enhancement are highly desirable to enhance the potentiality of SERS as a label-free optical tool for single molecule detection. Inspired by DNA origami technique, we constructed plasmonic dimer nanoantennas with a tunable gap decorated with Ag-coated Au nanostars on origami. Herein, we demonstrate the single-molecule SERS enhancements of three dyes with emission in different spectral regions after incorporation of single dye molecules in between two nanostars. The enhancement factors (EFs) achieved in the range of 109–1010 for all the single dye molecules, under both resonant and nonresonant excitation conditions, would enable enhanced photostability during time-series measurement. We further successfully explored the potential of our designed nanoantennas to accommodate and detect a single thrombin protein molecule after selective placement in the wide nanogap of 10 nm. Our results suggest that such nanoantennas can serve as a broadband SERS enhancer and enable specific detection of target biological molecules with single-molecule sensitivity.

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confidence: 99%

Broadband SERS Enhancement by DNA Origami Assembled Bimetallic Nanoantennas with Label-Free Single Protein Sensing

Tanwar¹,

Kaur²,

Kaur³

et al. 2021

J. Phys. Chem. Lett.

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“…Initial fully atomistic molecular dynamics (MD) simulations performed on the ligand-free form of thrombin in explicit water highlighted the dynamic behavior of the protein that was shown to be able to switch between an open state, likely related to the “fast form”, and a more compact conformation, possibly related to the “slow form” [ 130 ]. The remarkable dynamic propensity of thrombin and its correlation to the activity have been corroborated by later studies that have unraveled conformational states that were not present in the ensemble of the crystallographic structures [ 131 ]. In addition, these studies have underscored functional correlated motions between the active site and distant protein regions [ 132 ].…”

Section: Beyond a Static View Of Thrombin: Functional And Structural Evidence Of Exosite Communicationmentioning

confidence: 83%

Exosite Binding in Thrombin: A Global Structural/Dynamic Overview of Complexes with Aptamers and Other Ligands

Troisi

Balasco

Autiero

et al. 2021

IJMS

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Thrombin is the key enzyme of the entire hemostatic process since it is able to exert both procoagulant and anticoagulant functions; therefore, it represents an attractive target for the developments of biomolecules with therapeutic potential. Thrombin can perform its many functional activities because of its ability to recognize a wide variety of substrates, inhibitors, and cofactors. These molecules frequently are bound to positively charged regions on the surface of protein called exosites. In this review, we carried out extensive analyses of the structural determinants of thrombin partnerships by surveying literature data as well as the structural content of the Protein Data Bank (PDB). In particular, we used the information collected on functional, natural, and synthetic molecular ligands to define the anatomy of the exosites and to quantify the interface area between thrombin and exosite ligands. In this framework, we reviewed in detail the specificity of thrombin binding to aptamers, a class of compounds with intriguing pharmaceutical properties. Although these compounds anchor to protein using conservative patterns on its surface, the present analysis highlights some interesting peculiarities. Moreover, the impact of thrombin binding aptamers in the elucidation of the cross-talk between the two distant exosites is illustrated. Collectively, the data and the work here reviewed may provide insights into the design of novel thrombin inhibitors.

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“…The scenario changes in the presence of the irreversible inhibitor and transition state analog H-D-Phe-Pro-Arg-CH 2 Cl (PPACK) that makes extensive contacts with the active site 2,24 . Binding of PPACK is known to increase the stability of thrombin against chemical 48 and thermal 49 denaturation, but this effect is significantly reduced for the I16T and D194A mutants (Fig. 5B).…”

Section: Resultsmentioning

confidence: 99%

Role of the I16-D194 ionic interaction in the trypsin fold

Stojanovski

Chen

Koester

et al. 2019

Sci Rep

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Activity in trypsin-like proteases is the result of proteolytic cleavage at R15 followed by an ionic interaction that ensues between the new N terminus of I16 and the side chain of the highly conserved D194. This mechanism of activation, first proposed by Huber and Bode, organizes the oxyanion hole and primary specificity pocket for substrate binding and catalysis. Using the clotting protease thrombin as a relevant model, we unravel contributions of the I16-D194 ionic interaction to Na+ binding, stability of the transition state and the allosteric E*-E equilibrium of the trypsin fold. The I16T mutation abolishes the I16-D194 interaction and compromises the architecture of the oxyanion hole. The D194A mutation also abrogates the I16-D194 interaction but, surprisingly, has no effect on the architecture of the oxyanion hole that remains intact through a new H-bond established between G43 and G193. In both mutants, loss of the I16-D194 ionic interaction compromises Na+ binding, reduces stability of the transition state, collapses the 215–217 segment into the primary specific pocket and abrogates the allosteric E*-E equilibrium in favor of a rigid conformation that binds ligand at the active site according to a simple lock-and-key mechanism. These findings refine the structural role of the I16-D194 ionic interaction in the Huber-Bode mechanism of activation and reveal a functional linkage with the allosteric properties of the trypsin fold like Na+ binding and the E*-E equilibrium.

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Structure and Behavior of Human α-Thrombin upon Ligand Recognition: Thermodynamic and Molecular Dynamics Studies

Cited by 8 publications

References 58 publications

Broadband SERS Enhancement by DNA Origami Assembled Bimetallic Nanoantennas with Label-Free Single Protein Sensing

Broadband SERS Enhancement by DNA Origami Assembled Bimetallic Nanoantennas with Label-Free Single Protein Sensing

Exosite Binding in Thrombin: A Global Structural/Dynamic Overview of Complexes with Aptamers and Other Ligands

Role of the I16-D194 ionic interaction in the trypsin fold

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