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

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

Characterization of Cetyltrimethylammonium Bromide/Hexanol Reverse Micelles by Experimentally Benchmarked Molecular Dynamics Simulations

Encapsulation of small molecules, proteins, and other macromolecules within the protective water core of reverse micelles is emerging as a powerful strategy for a variety of applications. The cationic surfactant cetyl­trimethyl­ammonium bromide (CTAB) in combination with hexanol as a cosurfactant is particularly useful in the context of solution NMR spectroscopy of encapsulated proteins. Small-angle X-ray and neutron scattering is employed to investigate the internal structure of the CTAB/hexanol reverse micelle particle under conditions appropriate for high-resolution NMR spectroscopy. The scattering profiles are used to benchmark extensive molecular dynamics simulations of this reverse micelle system and indicate that the parameters used in these simulations recapitulate experimental results. Scattering profiles and simulations indicate formation of homogeneous solutions of small approximately spherical reverse micelle particles at a water loading of 20 composed of ∼150 CTAB and 240 hexanol molecules. The 3000 waters comprising the reverse micelle core show a gradient of translational diffusion that reaches that of bulk water at the center. Rotational diffusion is slowed relative to bulk throughout the water core, with the greatest slowing near the CTAB headgroups. The 5 Å thick interfacial region of the micelle consists of overlapping layers of Br^– enriched water, CTAB headgroups, and hexanol hydroxyl groups, containing about one-third of the total water. This study employs well-parametrized MD simulations, X-ray and neutron scattering, and electrostatic theory to illuminate fundamental properties of CTAB/hexanol reverse micelle size, shape, partitioning, and water behavior