Development and applications of NMR relaxation methods to study protein and ligand dynamics.

This thesis involves the study of proteins. These molecules exist in many variants, and perform most of our bodily functions. This includes signaling (for example the presence of capsaicin, which makes food spicy), molecular transport (including oxygen), catalysis (including food digestion) and part of our body structure (such as muscles). So it is perhaps not too strange that an overwhelming fraction of the medicines used and developed today target different proteins. The building blocks of proteins are 20 different amino acids. These amino acids can be linked in any conceivable combination and length to chains. Each protein is defined by their unique chain of amino acids. During the creation of each chain, it spontaneously folds due to the electrochemical forces that are always present between amino acids. The result of this folding process is called the protein structure, which help proteins to perform their tasks. To fully understand protein function, knowledge of the protein structure needs to be complemented with knowledge of protein dynamics, the inherent flexibility, and movements of the structure. The dynamic properties of proteins is the focus of this thesis, and the pharmaceutical industry its likeliest area of application. In the thesis I studied three different proteins. Galectin-3 is a protein that binds to sugar molecules, which can be found both inside and outside of cells. Problems with this protein are connected to a variety of diseases including cancer, fibrosis, and various forms of dementia. The protein BRD4 helps to control protein production in cells. This gives it a large impact on cancer progression and several types of cardiovascular diseases. When it comes to Ubiquitin, one of its main purposes is to signal which proteins have served their purpose and are to be degraded and recycled. In the thesis I study Ubiquitin only indirectly, as I use it as a model system given the wealth of previously published data to compare my results with. The main experimental method that I used is called Nuclear Magnetic Resonance spectroscopy (NMR), which share the same underlying technology as Magnetic Resonance Imaging (MRI) used in hospitals. My work also utilises results from other experimental methods, as different methods often complement each other. Important methods include Isothermal Calorimetry (ITC), which allows for measurements of the energy difference when two molecules bind to each other. Molecular Dynamics (MD) simulations, are a form of computer calculations that studies protein dynamics and interactions in atomistic detail. X-ray crystallography, uses high energy X-ray beams to discover protein structures. A total of five articles are presented in this thesis. The topic of the first article is the protein BRD4. It is a very large protein, with an amino acid chain of up to 1362 residues long. BRD4 is considered to have a large potential as a target for cancer treatments, but its length makes it hard to study. In the article we look at the impact on the protein dynamics from restricting studies of BRD4 to protein fragments of different lengths. The second article is a study on the interaction between Galectin-3 and a small molecule (similar to a pharmaceutical) from the perspective of the small molecule. This is accomplished by directly measuring on the fluorine atoms located on the small molecule. The third article is a study of the impact on interaction between Galectin-3 and a small molecule in the presence of Dimethyl Sulfoxide (DMSO). The largest effect of DMSO is an increased viscosity, which makes the interaction slower. The fourth and fifth articles presents a method for faster measurements of protein dynamics using NMR, which is especially useful for unstable and low-concentration samples. My hope is that this thesis work helps us gain an improved understanding on how different parts of proteins affect each other and on how proteins interact with small molecules (especially regarding BRD4 and Galectin-3). In addition, I hope that this work has provided future scientists additional tools for continued studies of proteins and their dynamics

Activated Transcription of the Human Neuropeptide Y Gene in Differentiating SH-SY5Y Neuroblastoma Cells Is Dependent on Transcription Factors AP-1, AP-2Α, and NGFI

Activated transcription of the human neuropeptide Y gene ( NPY ) was investigated in SH-SY5Y neuroblastoma cells at the onset of sympathetic neuronal differentiation induced by 12- O -tetradecanoylphorbol 13-acetate (TPA) and serum or by nerve growth factor (NGF). As determined by transient expression, two NGF response elements (REs) were required for transcription induced by NGF in SH-SY5Y cells with stable expression of an exogenous NGF receptor TRK-A gene (SH-SY5Y/trk). TPA treatment in the presence of serum induced NPY transcription in both wild-type SH-SY5Y (SH-SY5Y/wt) and SH-SY5Y/trk cells. A TPA RE (TRE), overlapping the proximal NGF RE, was identified by expression of the v-Jun oncoprotein that enhanced NPY transcription. Suppression of TPA-induced NPY transcription was obtained by expression of a dominant negative Jun protein, selective protein kinase C inhibition, or introduction of a mutated TRE, whereas NGF-induced NPY transcription was inhibited to a lesser degree. The transcription factor AP-2Α was shown to bind cooperatively to the NPY promoter with either AP-1 or NGFI-A to the shared TRE and NGF RE and to the distal NGF RE, respectively. These results show that transcription factors AP-1, AP-2Α, and NGFI-A are involved in activated NPY transcription during the onset of neuronal differentiation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65864/1/j.1471-4159.1998.70051887.x.pd

Structural model of dodecameric heat-shock protein Hsp21:Flexible N-terminal arms interact with client proteins while C-terminal tails maintain the dodecamer and chaperone activity

Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IXVXI motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the IXVXI motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations

Remote CAN operations in Matlab over the Internet

This paper describes the implementation of a CAN server that acts as a CAN tool to a client. It can be used to monitor, observe and send messages to a distant CAN network over IEEE802.11b (Wave-LAN). The CAN server is controlled by one or several clients that can connect to it by TCP/IP. It is possible to send and receive CAN messages over Internet from a MATLAB environment since the client software is written in Java. The CAN server collects CAN messages and stores them into a ring buffer. The messages in the ring buffer are classified by their identifier and stored into a database. The CAN tool has been used in a demonstrative application example that consist of a remotely controlled wheelchair. In the example the wheelchair was programmed to run in a square. The positions obtained by odometric CAN messages are compared with the position from the navigation system onboard the wheelchair.Godkänd; 2004; 20060929 (ysko)</p

Cosolvent Dimethyl Sulfoxide Influences Protein-Ligand Binding Kinetics via Solvent Viscosity Effects : Revealing the Success Rate of Complex Formation Following Diffusive Protein-Ligand Encounter

Protein-ligand-exchange kinetics determines the duration of biochemical signals and consequently plays an important role in drug design. Binding studies commonly require solubilization of designed ligands in solvents such as dimethyl sulfoxide (DMSO), resulting in residual amounts of DMSO following titration of solubilized ligands into aqueous protein samples. Therefore, it is critical to establish whether DMSO influences protein-ligand binding. Here, we address the general and indirect effect of DMSO on protein-ligand binding caused by solvent viscosity, which is strongly dependent on the relative concentrations of DMSO and water. As a model system, we studied the binding of a drug-like ligand to the carbohydrate recognition domain of galectin-3 in the presence of variable amounts of DMSO. We used isothermal titration calorimetry to characterize binding thermodynamics and 15N NMR relaxation to monitor kinetics. The binding enthalpy is not affected, but we observe a subtle trend of increasingly unfavorable entropy of binding, and consequently decreased affinity, with increasing DMSO concentration. The increasing concentration of DMSO results in a reduced association rate of binding, while the dissociation rate is less affected. The observed association rate is inversely proportional to the viscosity of the DMSO-water mixture, as expected from theory, but significantly reduced from the diffusion-controlled limit. By comparing the viscosity dependence of the observed association rate with that of the theoretical diffusion-controlled association rate, we estimate the success rate of productive complex formation following an initial encounter of proteins and ligands, showing that only one out of several hundred binding “attempts” are successful

Rapid measurement of heteronuclear transverse relaxation rates using non-uniformly sampled R1ρ accordion experiments

Multidimensional, heteronuclear NMR relaxation methods are used extensively to characterize the dynamics of biological macromolecules. Acquisition of relaxation datasets on proteins typically requires significant measurement time, often several days. Accordion spectroscopy offers a powerful means to shorten relaxation rate measurements by encoding the “relaxation dimension” into the indirect evolution period in multidimensional experiments. Time savings can also be achieved by non-uniform sampling (NUS) of multidimensional NMR data, which is used increasingly to improve spectral resolution or increase sensitivity per unit time. However, NUS is not commonly implemented in relaxation experiments, because most reconstruction algorithms are inherently nonlinear, leading to problems when estimating signal intensities, relaxation rate constants and their error bounds. We have previously shown how to avoid these shortcomings by combining accordion spectroscopy with NUS, followed by data reconstruction using sparse exponential mode analysis, thereby achieving a dramatic decrease in the total length of longitudinal relaxation experiments. Here, we present the corresponding transverse relaxation experiment, taking into account the special considerations required for its successful implementation in the framework of the accordion-NUS approach. We attain the highest possible precision in the relaxation rate constants by optimizing the NUS scheme with respect to the Cramér-Rao lower bound of the variance of the estimated parameter, given the total number of sampling points and the spectrum-specific signal characteristics. The resulting accordion-NUS R1ρ relaxation experiment achieves comparable precision in the parameter estimates compared to conventional CPMG (Carr-Purcell-Meiboom-Gill) R2 or spin-lock R1ρ experiments while saving an order of magnitude in experiment time

snowBOTS: a mobile robot on snow covered ice

We introduce snowBOTs as a generic name for robots working in snow. This paper is a study on using scan ning range measuring lasers towards an autonomous snow cleaning robot, working in an environment consisting al most entirely of snow and ice. The problem addressed here is using lasers for detecting the edges generated by "the snow meeting the road". First the laser data were filtered using his togram/median to discriminate against falling snowflakes and small objects. Then the road surface was extracted us ing the range weighted Hough/Radon transform. Finally the left and right edges of the road was detected by thresh olding. Tests have been made with a laser on top of a car driven in an automobile test range just south of the Arctic Circle. Moreover, in the campus area, the algorithms were tested in closed loop with the laser on board a robotized wheelchair.Validerad; 2007; 20070910 (hf)CASTT - Centre for Automotive Systems Technologies and Testin

An autonomous vehicle for a RobotDay

This paper describes the autonomous car that is being built for the RobotDay competition, arranged by SICK GmbH. The basic rule is to let an autonomous vehicle drive a track as fast as possible, based on any sensor technology. For this race SICK donated one S300 professional proximity sensor to each team. Our vehicle will use the circle sector expansion (CSE) method to avoid obstacles and to find its way along the track. We show initial results where the CSE method was used to guide the vehicle through a test track defined by cones and other objects.Godkänd; 2007; 20071005 (kalevi)CASTT - Centre for Automotive Systems Technologies and Testin

Backbone 1 H, 13 C, and 15 N resonance assignments of BoMan26A, a β-mannanase of the glycoside hydrolase family 26 from the human gut bacterium Bacteroides ovatus

Bacteroides ovatus is a member of the human gut microbiota. The importance of this microbial consortium involves the degradation of complex dietary glycans mainly conferred by glycoside hydrolases. In this study we focus on one such catabolic glycoside hydrolase from B. ovatus. The enzyme, termed BoMan26A, is a β-mannanase that takes part in the hydrolytic degradation of galactomannans. The crystal structure of BoMan26A has previously been determined to reveal a TIM-barrel like fold, but the relation between the protein structure and the mode of substrate processing has not yet been studied. Here we report residue-specific assignments for 95% of the 344 backbone amides of BoMan26A. The assignments form the basis for future studies of the relationship between substrate interactions and protein dynamics. In particular, the potential role of loops adjacent to glycan binding sites is of interest for such studies