Kinetics Of Structural Transitions Research Articles

T-Cell Receptor Variable \u03b2 Domains Rigidify During Affinity Maturation

We investigated T-cell receptor variable β chains binding to the superantigen staphylococcal enterotoxin C3 (SEC 3) with structure information in different stages of affinity maturation. Metadynamics in combination with molecular dynamics simulations allow to access the micro-to-millisecond timescale and reveal a strong effect of energetically significant mutations on the flexibility of the antigen-binding site. The observed changes in dynamics of the complementarity determining region (CDR) loops, especially the CDR 2, and HV 4 loop on this specific pathway of affinity maturation are reflected in their structural diversity, thermodynamics of conformations and kinetics of structural transitions. In addition, this affinity maturation pathway follows the concept of conformational selection, because even without the presence of the antigen the binding competent state is present in this pre-existing ensemble of conformations. In all stages of this affinity maturation process we observe a link between specificity and reduced flexibility.

Structural transition kinetics and activated behavior in the superconducting vortex lattice

Using small-angle neutron scattering, we investigated the behavior of a metastable vortex lattice state in MgB2 as it is driven towards equilibrium by an AC magnetic field. This shows an activated behavior, where the AC field amplitude and cycle count are equivalent to, respectively, an effective "temperature" and "time". The activation barrier increases as the metastable state is suppressed, corresponding to an aging of the vortex lattice. Furthermore, we find a cross-over from a partial to a complete suppression of metastable domains depending on the AC field amplitude, which may empirically be described by a single free parameter. This represents a novel kind of collective vortex behavior, most likely governed by the nucleation and growth of equilibrium vortex lattice domains.

Characterizing the Diversity of the CDR-H3 Loop Conformational Ensembles in Relationship to Antibody Binding Properties.

We present an approach to assess antibody CDR-H3 loops according to their dynamic properties using molecular dynamics simulations. We selected six antibodies in three pairs differing substantially in their individual promiscuity respectively specificity. For two pairs of antibodies crystal structures are available in different states of maturation and used as starting structures for the analyses. For a third pair we chose two antibody CDR sequences obtained from a synthetic library and predicted the respective structures. For all three pairs of antibodies we performed metadynamics simulations to overcome the limitations in conformational sampling imposed by high energy barriers. Additionally, we used classic molecular dynamics simulations to describe nano- to microsecond flexibility and to estimate up to millisecond kinetics of captured conformational transitions. The methodology represents the antibodies as conformational ensembles and allows comprehensive analysis of structural diversity, thermodynamics of conformations and kinetics of structural transitions. Referring to the concept of conformational selection we investigated the link between promiscuity and flexibility of the antibodies' binding interfaces. The obtained detailed characterization of the binding interface clearly indicates a link between structural flexibility and binding promiscuity for this set of antibodies.

Computational Approaches to Understanding the Biological Behaviour of Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) represent a class of proteins that lack a persistent folded conformation and exist as dynamic ensembles in their native state. Inherent lack of a well-defined structure and remarkable structural plasticity have facilitated their functioning in a wide range of crucial cellular processes such as signalling transduction and cell cycle regulation as well as responsible for their aberrant toxic amyloidogenic conformations implicated in a wide range of neurodegenerative diseases, cancer, etc. Their ubiquitous presence in nature, role in biological function and diseases have spurred interest in the biophysical and conformational characterization of IDPs. Conventional methods of structure determination are less feasible owing to structural and spatiotemporal heterogeneity of IDPs, which demand the development of novel biophysical methods as well as rigorous computational techniques for their characterization. In this review, we provide a brief overview of the widely used computational techniques probing the rugged conformational energy landscape of IDPs, their kinetics of structural transitions and molecular interactions key to their functions. Advances in the development of calibrated computational approaches for statistical representation of highly dynamic structural ensemble of IDPs are provided with examples. Challenges in modelling this unique class of proteins as well as the existing and futuristic avenues are also discussed.

FRET study of the structural and kinetic effects of PKC phosphomimetic cardiac troponin T mutants on thin filament regulation

FRET was used to investigate the structural and kinetic effects that PKC phosphorylations exert on Ca2+ and myosin subfragment-1 dependent conformational transitions of the cardiac thin filament. PKC phosphorylations of cTnT were mimicked by glutamate substitution. Ca2+ and S1-induced distance changes between the central linker of cTnC and the switch region of cTnI (cTnI-Sr) were monitored in reconstituted thin filaments using steady state and time resolved FRET, while kinetics of structural transitions were determined using stopped flow. Thin filament Ca2+ sensitivity was found to be significantly blunted by the presence of the cTnT(T204E) mutant, whereas pseudo-phosphorylation at additional sites increased the Ca2+-sensitivity. The rate of Ca2+-dissociation induced structural changes was decreased in the C-terminal end of cTnI-Sr in the presence of pseudo-phosphorylations while remaining unchanged at the N-terminal end of this region. Additionally, the distance between cTnI-Sr and cTnC was decreased significantly for the triple and quadruple phosphomimetic mutants cTnT(T195E/S199E/T204E) and cTnT(T195E/S199E/T204E/T285E), which correlated with the Ca2+-sensitivity increase seen in these same mutants. We conclude that significant changes in thin filament Ca2+-sensitivity, structure and kinetics are brought about through PKC phosphorylation of cTnT. These changes can either decrease or increase Ca2+-sensitivity and likely play an important role in cardiac regulation.

Structure-Function Studies of Loop L5 in the Mitotic Kinesin Eg5

All processive kinesins contain an unusual structural motif consisting of an alpha helix (α2) interrupted in the middle by a stem and loop motif known as L5. The role of L5 in the overall enzymatic function of kinesin motors remains unknown. However, its importance is highlighted by the finding that a variety of small molecule inhibitors of the mitotic kinesin Eg5, which contains the longest L5 in the kinesin superfamily, bind to this region with high affinity. These inhibitors induce a folding of L5, and trap the motor in an ADP bound, weak microtubule binding conformation. In order to gain greater insight into the function of L5, we have characterized three site-directed mutants in the loop (position 121), stem (position 131) or in the portion of α2 amino-terminal to this stem and loop motif (position 113). These three mutations have profound effects on the kinetics of structural transitions that occur with nucleotide and microtubule binding, and our results indicate that this conserved structural motif plays an important role in tuning the kinetics of kinesin motors.

Structural transition of the inhibitory region of troponin I within the regulated cardiac thin filament

Contraction and relaxation of cardiac muscle are regulated by the inhibitory and regulatory regions of troponin I (cTnI). Our previous FRET studies showed that the inhibitory region of cTnI in isolated troponin experiences a structural transition from a β-turn/coil motif to an extended conformation upon Ca 2+ activation. During the relaxation process, the kinetics of the reversal of this conformation is coupled to the closing of the Ca 2+-induced open conformation of the N-domain of troponin C (cTnC) and an interaction between cTnC and cTnI in their interface. We have since extended the structural kinetic study of the inhibitory region to fully regulated thin filament. Single-tryptophan and single-cysteine mutant cTnI(L129W/S151C) was labeled with 1,5-IAEDANS at Cys151, and the tryptophan-AEDANS pair served as a donor–acceptor pair. Labeled cTnI mutant was used to prepare regulated thin filaments. Ca 2+-induced conformational changes in the segment of Trp129-Cys151 of cTnI were monitored by FRET sensitized acceptor (AEDANS) emission in Ca 2+ titration and stopped-flow measurements. Control experiments suggested energy transfer from endogenous tryptophan residues of actin and myosin S1 to AEDANS attached to Cys151 of cTnI was very small and Ca 2+ independent. The present results show that the rate of Ca 2+-induced structural transition and Ca 2+ sensitivity of the inhibitory region of cTnI were modified by (1) thin filament formation, (2) the presence of strongly bound S1, and (3) PKA phosphorylation of the N-terminus of cTnI. Ca 2+ sensitivity was not significantly changed by the presence of cTm and actin. However, the cTn–cTm interaction decreased the cooperativity and kinetics of the structural transition within cTnI, while actin filaments elicited opposite effects. The strongly bound S1 significantly increased the Ca 2+ sensitivity and slowed down the kinetics of structural transition. In contrast, PKA phosphorylation of cTnI decreased the Ca 2+ sensitivity and accelerated the structural transition rate of the inhibitory region of cTnI on thin filaments. These results support the idea of a feedback mechanism by strong cross-bridge interaction with actin and provide insights on the molecular basis for the fine tuning of cardiac function by β-adrenergic stimulation.

Structural Transitions of Confined Model Proteins: Molecular Dynamics Simulation and Experimental Validation

Proteins fold in a confined space not only in vivo, i.e., folding assisted by molecular chaperons and chaperonins in a crowded cellular medium, but also in vitro as in production of recombinant proteins. Despite extensive work on protein folding in bulk, little is known about how and to what extent the thermodynamics and kinetics of protein folding are altered by confinement. In this work, we use a Gō-like off-lattice model to investigate the folding and stability of an all β-sheet protein in spherical cages of different sizes and surface hydrophobicity. We find whereas extreme confinement inhibits correct folding, a hydrophilic cage stabilizes the protein due to restriction of the unfolded configurations. In a hydrophobic cage, however, strong attraction from the cage surface destabilizes the confined protein because of competition between self-aggregation and adsorption of hydrophobic residues. We show that the kinetics of protein collapse and folding is strongly correlated with both the cage size and the surface hydrophobicity. It is demonstrated that a cage of moderate size and hydrophobicity optimizes both the folding yield and kinetics of structural transitions. To support the simulation results, we have also investigated the refolding of hen-egg lysozyme in the presence of cetyltrimethylammoniumbromide (CTAB) surfactants that provide an effective confinement of the proteins by micellization. The influence of the surfactant hydrophobicity on the structural and biological activity of the protein is determined with circular dichroism spectrum, fluorescence emission spectrum, and biological activity assay. It is shown that, as predicted by coarse-grained simulations, CTAB micelles facilitate the collapse of denatured lysozyme, whereas the addition of β-cyclodextrin-grafted-PNIPAAm, a weakly hydrophobic stripper, dissociates CTAB micelles and promotes the conformational rearrangement and thereby gives an improved recovery of lysozyme activity.

時間分割蛍光エネルギー移動法による細いフィラメント上の構造的な動きの速度論

時間分割蛍光エネルギー移動法による細いフィラメント上の構造的な動きの速度論

Micelle-to-Vesicle Transition: A Time-Resolved Structural Study

Amphiphilic molecules spontaneously self-assemble in solution into a variety of microstructures. While their equilibrium properties are well understood, little is known about the kinetics of structural transitions and the existence and properties of metastable states. We demonstrate for the micelle-to-vesicle transition that the evolution of aggregate structures can be followed using time-resolved light and neutron scattering. The polymerlike micelles are found to first transform into nonequilibrium disks, which finally evolve into vesicles following an exponential time dependence.

Kinetics of structural transitions in surfactant solutions

While the equilibrium behaviour of surfactant solutions is well studied, the understanding of the kinetics and pathways of structural transitions under nonequilibrium conditions is only beginning to develop. Attention has recently been directed mainly towards micellar kinetics, transitions between micellar and lamellar phases, vesicle fusion, and phase separation in microemulsions. This progress has profited greatly from developments that have taken place in various techniques and instruments.

On the kinetics of structural transition I of some pancreatic proteins

On the kinetics of structural transition I of some pancreatic proteins