18 research outputs found
The Dynamic Structure of Thrombin in Solution
AbstractThe backbone dynamics of human α-thrombin inhibited at the active site serine were analyzed using R1, R2, and heteronuclear NOE experiments, variable temperature TROSY 2D [1H-15N] correlation spectra, and Rex measurements. The N-terminus of the heavy chain, which is formed upon zymogen activation and inserts into the protein core, is highly ordered, as is much of the double beta-barrel core. Some of the surface loops, by contrast, remain very dynamic with order parameters as low as 0.5 indicating significant motions on the ps-ns timescale. Regions of the protein that were thought to be dynamic in the zymogen and to become rigid upon activation, in particular the γ-loop, the 180s loop, and the Na+ binding site have order parameters below 0.8. Significant Rex was observed in most of the γ-loop, in regions proximal to the light chain, and in the β-sheet core. Accelerated molecular dynamics simulations yielded a molecular ensemble consistent with measured residual dipolar couplings that revealed dynamic motions up to milliseconds. Several regions, including the light chain and two proximal loops, did not appear highly dynamic on the ps-ns timescale, but had significant motions on slower timescales
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Exploring the Dynamics of Thrombin by NMR
Thrombin is a tightly regulated serine protease that acts at the terminus of the blood coagulation cascade, performing proteolytic activation of platelets and converting soluble fibrinogen into insoluble fibrin. In complex with thrombomodulin, thrombin acts as an anticoagulant by activating protein C. The strict regulation of thrombin is essential to organism survival, as under-regulation of clot formation results in amplified blood loss and over-regulation leads to pathological clot formation. In human health, hemophilia, pulmonary emboli, venous thrombi, myocardial infarctions and ischemic strokes are all tied to the misregulation of thrombin. Structurally, thrombin consists of a double [Beta]-barrel core that is conserved among members of the trypsin-like protease family with extended active site loops that are not conserved. Recent evidence has suggested that the regulation of thrombin is tied much more to dynamics than previously thought and NMR is the ideal method of study. Chapter II outlines the process of isotopic labeling, expression, refolding, activation and purification of recombinant thrombin from E. coli. Once this barrier was overcome, the next hurdle in the study of thrombin by NMR was resonance assignments, discussed in Chapter III. Results from resonance assignment of thrombin with PPACK occupying its active site vs. the S195M mutant representing apo-thrombin showed major chemical shift perturbations between the forms and a large degree of resonance line broadening that abrogated the signal, an effect that was mostly abolished upon active site ligation by PPACK. In Chapter IV, using a combined NMR and computational approach (performed by P. Gasper and P. Markwick of the McCammon lab) the dynamics of PPACK- thrombin are characterized and the solution ensemble is modeled. The results indicate that even with the active site occupied by PPACK, thrombin undergoes a large degree of structural fluctuations on timescales ranging from picoseconds all the way to milliseconds. The equivalent study was undertaken on S195M-thrombin as discussed in Chapter V. As compared to PPACK-thrombin, the apo-like S195M-thrombin, apo-thrombin displays a remarkable degree of dynamics in the [mu]s-ms timescale. These results reinforce the paradigm shift towards an ensemble view of thrombin activity states and substrate recognitio
Exploring the Dynamics of Thrombin by NMR
Thrombin is a tightly regulated serine protease that acts at the terminus of the blood coagulation cascade, performing proteolytic activation of platelets and converting soluble fibrinogen into insoluble fibrin. In complex with thrombomodulin, thrombin acts as an anticoagulant by activating protein C. The strict regulation of thrombin is essential to organism survival, as under-regulation of clot formation results in amplified blood loss and over-regulation leads to pathological clot formation. In human health, hemophilia, pulmonary emboli, venous thrombi, myocardial infarctions and ischemic strokes are all tied to the misregulation of thrombin. Structurally, thrombin consists of a double [Beta]-barrel core that is conserved among members of the trypsin-like protease family with extended active site loops that are not conserved. Recent evidence has suggested that the regulation of thrombin is tied much more to dynamics than previously thought and NMR is the ideal method of study. Chapter II outlines the process of isotopic labeling, expression, refolding, activation and purification of recombinant thrombin from E. coli. Once this barrier was overcome, the next hurdle in the study of thrombin by NMR was resonance assignments, discussed in Chapter III. Results from resonance assignment of thrombin with PPACK occupying its active site vs. the S195M mutant representing apo-thrombin showed major chemical shift perturbations between the forms and a large degree of resonance line broadening that abrogated the signal, an effect that was mostly abolished upon active site ligation by PPACK. In Chapter IV, using a combined NMR and computational approach (performed by P. Gasper and P. Markwick of the McCammon lab) the dynamics of PPACK- thrombin are characterized and the solution ensemble is modeled. The results indicate that even with the active site occupied by PPACK, thrombin undergoes a large degree of structural fluctuations on timescales ranging from picoseconds all the way to milliseconds. The equivalent study was undertaken on S195M-thrombin as discussed in Chapter V. As compared to PPACK-thrombin, the apo-like S195M-thrombin, apo-thrombin displays a remarkable degree of dynamics in the [mu]s-ms timescale. These results reinforce the paradigm shift towards an ensemble view of thrombin activity states and substrate recognitio
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Correlated motions and residual frustration in thrombin.
Thrombin is the central protease in the cascade of blood coagulation proteases. The structure of thrombin consists of a double β-barrel core surrounded by connecting loops and helices. Compared to chymotrypsin, thrombin has more extended loops that are thought to have arisen from insertions in the serine protease that evolved to impart greater specificity. Previous experiments showed thermodynamic coupling between ligand binding at the active site and distal exosites. We present a combined approach of molecular dynamics (MD), accelerated molecular dynamics (AMD), and analysis of the residual local frustration of apo-thrombin and active-site-bound (PPACK-thrombin). Community analysis of the MD ensembles identified changes upon active site occupation in groups of residues linked through correlated motions and physical contacts. AMD simulations, calibrated on measured residual dipolar couplings, reveal that upon active site ligation, correlated loop motions are quenched, but new ones connecting the active site with distal sites where allosteric regulators bind emerge. Residual local frustration analysis reveals a striking correlation between frustrated contacts and regions undergoing slow time scale dynamics. The results elucidate a motional network that probably evolved through retention of frustrated contacts to provide facile conversion between ensembles of states