Novel weak alignment techniques for nuclear magnetic resonance spectroscopy and applications to biomolecular structure determination

Nuclear magnetic resonance spectroscopy has continuously been developing ever since its introduction as a structural method in bioscience. Recently established residual dipolar coupling techniques yield information on long-range order in weakly aligned samples as they define the orientation of vectors between nuclei in a common global reference frame. These data complement classical short-range information and have a unique potential especially for the characterization of non-globular states. This thesis describes the development of novel methods for the weak alignment of biomacromolecules in charged gels and for the measurement of long-range residual dipolar couplings in perdeuterated proteins. These weak alignment techniques and other nuclear magnetic resonance methods were applied to study the structure and folding of various proteins such as the fibritin folding nucleus, the minicollagen cysteine rich domain and human protein tyrosine phosphatase 1B

All things hydrogen: hydrogen imaging in atom probe tomography and its application to tungsten

This thesis investigates the fundamental behaviour of contaminant hydrogen in Atom Probe Tomography (APT), develops an APT imaging method for sample hydrogen, applies it to deuterium implanted tungsten, and describes a large-scale APT data analysis and its application to hydrogen. Rate cycling experiments are introduced, where the evaporation rate or frequency is repeatedly changed during experiments. Contaminant hydrogen is, other than sample hydrogen, rate dependent, therefore this allows estimating the true amount of sample hydrogen. By undertaking APT experiments below the evaporation threshold, it could then be investigated how the electric field influences the contaminant signal on tungsten. Beyond a critical field strength, the contaminants are found to evaporate with a detectable energy deficit. This allows differentiation between contaminant and sample hydrogen, which evaporates without any energy deficit, enabling development of a method for direct hydrogen imaging using pulsed laser APT. The technique is applied to image deuterium in self-ion irradiated, deuterium implanted tungsten. The deuterium is retained with a characteristic depth distribution, which is imaged by APT with high spatial resolution. The highest deuterium concentrations are found in a layer directly under the material surface (up to 20 nm deep), corresponding to the depth reached by the deuterium during implantation, and in a deeper layer (ca 120 to 170 nm below) due to radiation damage. The scatter of measured concentrations across samples is large, and potentially requires further study. Finally, a large-scale data analysis, covering 848 archival datasets from the Oxford APT group is presented. It is shown that many APT spectra can be regarded as a point cloud, in which a similarity metric based on the city block distance is well suited to search spectra by similarity. The point cloud contains clusters, mostly due to chemical composition of the underlying APT specimens. The dependence of contaminant hydrogen behaviour on material, voltage and electric field strength is studied

Supramolecular chemical shift reagents inducing conformational transitions: NMR analysis of carbohydrate homooligomer mixtures

Supramolecular chemical shift reagents improve signal resolution for NMR analysis of homooligosaccharides by inducing conformational transitions upon binding.</p

Global expression mapping of mammalian genomes

he aim of genome projects is to decipher all the information contained within the DNA of an organism and to study the way this information is processed in physiological processes. It is believed that more than 95% of the information content of the mammalian genome is represented in the protein coding sequences that make up only approximately 2% of the DNA sequence. Consequently much effort is being invested in the study of coding sequences in the form of cDNA analysis. This thesis is concerned with the development of a new strategy for a highly parallel approach to analyse entire cDNA libraries. The strategy is based upon generating sufficient sequence information to identify uniquely more than 100,000 cDNA clones by hybridisation with short oligonucleotides, typically 7 - 10 mers. Each oligonucleotide is hybridised to all cDNA clones in parallel and under stringent conditions positively identifies a subset (3 - 10%) of clones. Oligonucleotides are designed in such a way that each will positively identify a different subset of clones and statistical simulations estimate that approximately 200 such hybridisation events are required to identify uniquely upto 100,000 cDNA sequences. Such a fingerprint can be generated from many cDNA libraries constructed from different tissue mRNAs and will not only lead to the identification of most sequecnes expressed from the genome but also indicate the level of expression by determining the number of times any given sequence is represented across different cDNA libraries. A human foetal brain cDNA library has been constructed and 100,000 clones arrayed into microtitre plates and on nylon membranes. All the required technological developments have been carried out successfully and are presented. In excess of 200 oligonucleotide hybridisations have been performed on a subset of 32,000 cDNA clones and 1,000 sequenced control clones. A detailed analysis of the data on the control clones is presented and the implications for cDNA fingerprinting discussed

¹H-¹³C NMR-based profiling of biotechnological starch utilization

Starch is used in food- and nonfood applications as a renewable and degradable source of carbon and energy. Insight into the chemical detail of starch degradation remains challenging as the starch constituents amylose and amylopectin are homopolymers. We show that considerable molecular detail of starch fragmentation can be obtained from multivariate analysis of spectral features in optimized ¹H–¹³C NMR spectroscopy of starch fragments to identify relevant features that distinguish processes in starch utilization. As a case study, we compare the profiles of starch fragments in commercial beer samples. Spectroscopic profiles of homooligomeric starch fragments can be excellent indicators of process conditions. In addition, differences in the structure and composition of starch fragments have predictive value for downstream process output such as ethanol production from starch. Thus, high-resolution ¹H–¹³C NMR spectroscopic profiles of homooligomeric fragment mixtures in conjunction with chemometric methods provide a useful addition to the analytical chemistry toolbox of biotechnological starch utilization

Spectroscopic approaches to resolving ambiguities of hyper-polarized NMR signals from different reaction cascades

Ambiguities in identifying transient intracellular reaction intermediates are resolved by site-specific isotope labelling, optimised referencing and response to external perturbations.</p

Hyperpolarised Organic Phosphates as NMR Reporters of Compartmental pH

When formed in defined cellular compartments from exogenous hyperpolarised13C substrates, metabolites yield correlations of compartmental pH and catalytic activity.</p

Charged acrylamide copolymer gels as media for weak alignment

The use of mechanically strained acrylamide/acrylate copolymers is reported as a new alignment medium for biomacromolecules. Compared to uncharged, strained polyacrylamide gels, the negative charges of the acrylamide/acrylate copolymer strongly alter the alignment tensor and lead to pronounced electroosmotic swelling. The swelling itself can be used to achieve anisotropic, mechanical strain. The method is demonstrated for the alignment of TipAS, a 17kDa antibiotic resistance protein, as well as for human ubiquitin, where alignment tensors with an AZZ,NH of up to 60Hz are achieved at a gel concentration of 2% (w/v). The alignment can be modulated by the variation of pH, ionic strength, and gel concentration. The high mechanical stability of the swollen gels makes it possible to obtain alignment at polymer concentrations of less than 1% (w/v