Human thiopurine methyltransferase (TPMT) is an enzyme involved in the metabolism of thiopurine drugs, which are widely used in leukemia and inflammatory bowel diseases such as ulcerative colitis and Crohn´s disease. Due to genetic polymorphisms, approximately 30 protein variants are present in the population, some of which have significantly lowered activity. TPMT *5 (Leu49Ser) is one of the protein variants with almost no activity. The mutation is positioned in the hydrophobic core of the protein, close to the active site. Hydrogen exchange rates measured with NMR spectroscopy for N-terminally truncated constructs of TPMT *5 and TPMT *1 (wild type) show that local stability and hydrogen bonding patterns are changed by the mutation Leu49Ser. Most residues exhibit faster exchange rates and a lower local stability in TPMT *5 in comparison with TPMT *1. Changes occur close to the active site but also throughout the entire protein. Calculated overall stability is similar for the two constructs, so the measured changes are due to local stability. Protein dynamics measured with NMR relaxation experiments show that both TPMT *5 and TPMT *1 are monomeric in solution. Millisecond dynamics exist in TPMT *1 but not in TPMT *5, even though a few residues exhibit a faster dynamic. Dynamics on nanosecond to picosecond time scale have changed but no clear trends are observable.

none

Two multiscale continuum models for simulating protein dynamics are developed which allow for resolution of protein peptide planes in a beam-like finite element. A curvature and strain based finite element formulation is utilized. This formulation is advantageous in simulating proteins since amino acid chains may be described by a single element, even when the protein segment considered exhibits large curvature and twist such as the alpha-helical shapes prominent in many proteins. Specifically, concurrent and hierarchical multiscale models are developed for the curvature and strain based beam formulation. The hierarchical multiscale continuum model utilizes a novel shooting method to calculate the deformed configuration of the protein. An optimization algorithm determines the requisite stiffness parameters by varying the beam stiffness used in the shooting method until deformed configurations of test cases correspond to those produced by the LAMMPS molecular dynamics software. Additionally, a concurrent multiscale method is detailed for evaluating protein inter-atomic potential parameters from the curvature and strain degrees of freedom employed in the model. This allows internal forces and moments to be calculated using nonlinear protein potentials. Proof of concept testing and model verification for both models includes comparing the multiscale techniques to all-atom molecular dynamics solutions. Specifically, the models are verified by simulating a polypeptide in a vacuum and comparing the predicted results to those computed using LAMMPS.

Coupling the small GTPase Rab3 to the Synaptic Vesicle Cycle

No description available.

The topology and dynamics of biomolecules are intricately linked with their biological function. The focus of this thesis is the NMR-based measurement of topology and dynamics in biomolecular systems, and methods of measuring immersion depth and orientation of membrane-associated molecules. In detergent micelles and lipid bilayers, the local concentrations of hydrophobic and hydrophilic molecules are a function of their bilayer immersion depth. For paramagnetic molecular oxygen or metal cations, the magnitudes of the associated paramagnetic isotropic contact shifts and relaxation rate enhancements (PREs) are therefore depth-dependent. NMR measurements of these effects reveal the immersion depth of bilayer- or detergent-associated molecules. This work first explores transbilayer oxygen solubility and thermodynamics, as measured from contact shifts and PREs of the constituent lipid molecules in the presence of 30 bar oxygen. Contact shifts revealed the transmembrane O2 solubility profile spans a factor of seven across the bilayer, while PREs indicated that oxygen partitioning into bilayers and dodecylphosphocholine (DPC) micelles is entropically driven. Next, this work describes how paramagnetic effects from molecular oxygen and Ni(II) cations may be employed to study the immersion depth and topology of drug and protein molecules in DPC micelles. In one study, the positioning of the amphipathic drug imipramine in micelles was determined from O2- and Ni(II)-induced contact shifts. A second study, relying solely on O2-induced PREs, determined the tilt angles and micelle immersion depths of the two alpha helices in a monomeric mutant of the membrane protein phospholamban. A third study utilized 19F NMR to explore the importance of juxtamembraneous tryptophans on the topology of the membrane protein synaptobrevin, via O2-induced contact shifts and solvent-induced isotope shifts of a juxtamembraneous 19F-phenylalanine. Comparison of synaptobrevin constructs with zero, one, and two juxtamembraneous tryptophans revealed that while one tryptophan is sufficient to ‘anchor’ the protein in micelle, the addition of a second tryptophan dampens local dynamics. These solution state NMR studies demonstrate how paramagnetic effects from dissolved oxygen, complemented with measurements of local water exposure, provide detailed, accurate descriptions of membrane immersion depth and topology. These techniques are readily extended to the study of a wide range of biomolecules.

Each member of the protein kinase C (PKC) family activates cell signaling pathways with different and sometimes opposing cell functions, such as cell division, migration, or death. Because of the importance of these processes in human diseases and disorders like cancer, stroke, and Alzheimer’s disease, there is a need for drugs which modify the action of PKC. However, drug design is difficult due to the complicated nature of PKC regulation. To better understand the differential regulation of PKC activity, these studies probe the structure, dynamics, and reactivity of one of the domains responsible for PKC regulation, C1B. C1B binds signaling molecules and translocates PKC to membranes in order to release the kinase domain from inhibition. Mutagenesis and ligand-binding assays monitored with fluorescence and nuclear magnetic resonance (NMR) techniques show that a single variable residue in C1B dramatically affects the sensitivity to signal activators. Investigation of the domain structure and dynamics using NMR revealed the identity of this residue alters the dynamics of the activator binding loops, without changing the structure. NMR studies of the C1B variants in membrane-mimicking micelles showed this residue also changes the interaction of the regulatory domain with lipids. These results demonstrate PKC isoforms have evolved specific functions by tuning dynamics and membrane affinity. Alternatively, PKC can be activated by reactive oxygen species by a mechanism that does not require binding of signaling molecules or membrane localization. To investigate the role of C1B in this type of signaling, the regulatory domain reactivity is monitored via NMR and gel electrophoresis. These studies reveal a particular cysteine residue in C1B that is most reactive, an alternative conformation of C1B in which this residue is more exposed, and modification of C1B leads to unfolding and zinc loss. Because the regulatory domains are responsible for auto-inhibition of the kinase domain, C1B unfolding provides a plausible explanation for activation of PKC by reactive oxygen species. The relation of the intrinsic C1B properties to the activation of PKC can be used to develop drugs with a single mechanism and to better understand how closely related signaling proteins develop specific functions.

The topology and dynamics of biomolecules are intricately linked with their biological function. The focus of this thesis is the NMR-based measurement of topology and dynamics in biomolecular systems, and methods of measuring immersion depth and orientation of membrane-associated molecules. In detergent micelles and lipid bilayers, the local concentrations of hydrophobic and hydrophilic molecules are a function of their bilayer immersion depth. For paramagnetic molecular oxygen or metal cations, the magnitudes of the associated paramagnetic isotropic contact shifts and relaxation rate enhancements (PREs) are therefore depth-dependent. NMR measurements of these effects reveal the immersion depth of bilayer- or detergent-associated molecules. This work first explores transbilayer oxygen solubility and thermodynamics, as measured from contact shifts and PREs of the constituent lipid molecules in the presence of 30 bar oxygen. Contact shifts revealed the transmembrane O2 solubility profile spans a factor of seven across the bilayer, while PREs indicated that oxygen partitioning into bilayers and dodecylphosphocholine (DPC) micelles is entropically driven. Next, this work describes how paramagnetic effects from molecular oxygen and Ni(II) cations may be employed to study the immersion depth and topology of drug and protein molecules in DPC micelles. In one study, the positioning of the amphipathic drug imipramine in micelles was determined from O2- and Ni(II)-induced contact shifts. A second study, relying solely on O2-induced PREs, determined the tilt angles and micelle immersion depths of the two alpha helices in a monomeric mutant of the membrane protein phospholamban. A third study utilized 19F NMR to explore the importance of juxtamembraneous tryptophans on the topology of the membrane protein synaptobrevin, via O2-induced contact shifts and solvent-induced isotope shifts of a juxtamembraneous 19F-phenylalanine. Comparison of synaptobrevin constructs with zero, one, and two juxtamembraneous tryptophans revealed that while one tryptophan is sufficient to ‘anchor’ the protein in micelle, the addition of a second tryptophan dampens local dynamics. These solution state NMR studies demonstrate how paramagnetic effects from dissolved oxygen, complemented with measurements of local water exposure, provide detailed, accurate descriptions of membrane immersion depth and topology. These techniques are readily extended to the study of a wide range of biomolecules.

Previous research has underscored the importance of protein dynamics during light-induced electron transfer; however, specific interactions have not been well characterized. It is of particular importance to understand the role of protein dynamics and cofactor interactions in controlling electron transfer in oxygenic photosynthesis. These factors include hydrogen bonding, ð-stacking and electrostatic interactions. Reaction-induced FT-IR spectroscopy is sensitive to these interactions as well as isotopic incorporation, and is useful to probe protein dynamics associated with light-induced electron transfer in Photosystem I (PSI). Density functional theory (DFT) provides information concerning the vibrational frequencies of molecules as well as the amplitudes of the vibrations and sensitivity to isotope incorporation. Combining these approaches, protein dynamics associated with light-induced electron transfer in PSI were studied. The work presented here describes specific protein cofactor interactions and specific protein relaxation events associated with light-induced electron transfer. The results reported here are consistent with noncovalent protein cofactor interactions that modulate the redox potential of the secondary electron acceptor of PSI. Furthermore, the studies presented here describe novel protein dynamics associated with the oxidation of the terminal electron donor of PSI. These results characterize specific protein dynamics that may be associated with interactions of the soluble electron donors. These studies highlight the importance of protein dynamics in oxygenic photosynthesis.

Biological molecules often undergo large structural changes to perform their function. Computational methods can provide a fine-grained description at the atomistic scale. Without sufficient approximations to accelerate the simulations, however, the time-scale on which functional motions often occur is out of reach for many traditional methods. Natural Move Monte Carlo belongs to a class of methods that were introduced to bridge this gap. I present three novel applications for Natural Move Monte Carlo, two on proteins and one on DNA epigenetics. In the second part of this thesis I introduce a new protocol for the testing of hypotheses regarding the functional motions of biological systems, named customised Natural Move Monte Carlo. Two different case studies are presented aimed at demonstrating the feasibility of customised Natural Move Monte Carlo.