The ability of mutations to change the fold of a protein provides evolutionary pathways to new structures. To study hypothetical pathways for protein fold evolution, we designed intermediate sequences between Xfaso1 and Pfl6, two homologous Cro proteins that have 40% sequence identity but adopt all–α and α+β folds, respectively. The designed hybrid sequences XPH1 and XPH2 have 70% sequence identity to each other. XPH1 is more similar in sequence to Xfaso1 (86% sequence identity) while XPH2 is more similar to Pfl6 (80% sequence identity). NMR solution ensembles show that XPH1 and XPH2 have structures intermediate between Xfaso1 and Pfl6. Specifically, XPH1 loses α-helices 5 and 6 of Xfaso1 and incorporates a small amount of β-sheet structure; XPH2 preserves most of the β-sheet of Pfl6 but gains a structure comparable to helix 6 of Xfaso1. These findings illustrate that the sequence space between two natural protein folds may encode a range of topologies, which may allow a protein to change its fold extensively through gradual, multistep mechanisms. Evolving a new fold may have consequences, such as a strained conformation. Here we show that Pfl6 represents an early, strained form of the α+β Cro fold resulting from an ancestral remnant of the all-α Cro proteins retained after the fold switch. This nascent fold can be stabilized through deletion mutations in evolution, which can relieve the strain but may also negatively affect DNA-binding function. Compensatory mutations that increase dimerization appear to offset these effects to maintain function. These findings suggest that new folds can undergo mutational editing through evolution, which may occur in parallel pathways with slightly different outcomes.

Inhibition of the intrinsically disordered proteins (IDP) is a recognized issue in drug research. Standard approaches, based on key-lock model, cannot be used in the absence of rigid structure and defined active site. Here a basic helix-loop-helix leucine zipper (bHLHZip) domain of c-Myc was studied, which is intrinsically disordered and prone to aggregation. Chemical denaturation of proteins is a widely accepted technique to study protein folding, but here this methodology was applied to IDP, observing its effect on the structural ensemble of c-Myc by NMR spectroscopy. Nonlinear chemical shift changes indicated cooperative unfolding of the helical structure of the part of the leucine zipper domain in parallel with the melting of the N-terminal helix. Paramagnetic relaxation enhancement (PRE) was used to probe long-range structure and revealed presence of long-range contacts. The following search for inhibitors can be directed to the search for ligands, locking c-Myc in its more compact conformation. Protein self-association is a problem typical for IDPs and intrinsic process for all proteins at high concentrations. It leads to increased viscosity, gelation and possible precipitation, which cause problems in protein manufacturing, stability and delivery. If protein drugs require high dosing, special approaches are needed. At high concentrations proteins experience conditions close to the crystal state, therefore interactions in solution could potentially coincide with crystal lattice contacts. A range of diverse methods is used to study this process, but the complexity of the mechanism makes it hard to build a reliable model. Here, the self-association of streptococcal Protein G (PrtG) was studied using Nuclear Magnetic Resonance (NMR) spectroscopy in solution. The properties of protein-protein interactions at high concentration, up to ~ 160 mg/ml, were studied at residue-level resolution. Residue specific information on protein dynamics was obtained using 15N relaxation measurements. The experiments were carried out at multiple concentrations. Variation in the rotational correlation time over these concentrations showed changes in the protein dynamics, which indicated weak protein-protein interactions occurring in solution. Pulsed-field gradient NMR spectroscopy was used to monitor translational diffusion coefficients in order to estimate the degree of protein self-association. Oligomer formation was also monitored by looking at variations in 1H and 15N amide chemical shifts. Better understanding of protein self-association mechanisms under different conditions could assist in developing methods to reduce the level of reversible protein self-association in solution at high protein concentrations.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19315号 / 工博第4112号 / 新制||工||1634(附属図書館) / 32317 / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 白川 昌宏, 教授 跡見 晴幸, 教授 梶 弘典 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Nuclear Magnetic Resonance (NMR) spectroscopy is a quantitative, non-invasive and non-destructive technique useful in biological studies. By manipulating the magnetization of nuclei with non-zero spin, NMR gives insights into atomic level details. Application of NMR as a tool for discovering structure, understanding dynamics of bio-molecules such as proteins, metabolites, DNA, RNA and their interactions constitutes the field of bio-molecular NMR. In this thesis, new methods for rapid data analysis of NMR spectrum of proteins and metabolites are proposed. The first computational method, PROMEB (Pattern Recognition Based Assignment in Metabolomics) is useful for the identification and assignments of metabolites. This is an important step in metabolomics and is necessary for the discovery of new biomarkers. In NMR spectroscopy based studies, the conventional approach involves a database search, wherein chemical shifts are assigned to specific metabolites by use of a tolerance limit. This is inefficient because deviation in chemical shifts associated with pH or temperature variations, as well as missing peaks, impairs a robust comparison with the database. These drawbacks are overcome in PROMEB, which is a method based on matching the pattern of peaks of a metabolite in 2D [13C, 1H] HSQC NMR spectrum, rather than conventionally used absolute tolerance thresholds. A high success rate is obtained even in the presence of large chemical shift deviations such as 0.5 ppm in 1H and 3 ppm in 13C and missing peaks (up to 50%), compared to nearly no assignments obtained under these conditions with existing methods that employ a direct database search approach. The pattern recognition approach thus helps in identification and assignment of metabolites in-dependent of the pH, temperature, and ionic strength used, thereby obviating the need for spectral calibration with internal or external standards. Another computational method, ChemSMP(Chemical Shifts to Metabolic Path-ways), is described which facilitates the identification of metabolic pathways from a single two dimensional (2D) NMR spectrum. Typically in other approaches, this is done after relevant metabolites are identified to allow their mapping onto specific metabolic pathways. This task is daunting due to the complex nature of cellular processes and the difficulty in establishing the identity of individual metabolites. ChemSMP uses a novel indexing and scoring system comprised of a uniqueness score and a coverage score. Benchmarks show that ChemSMP has a positive prediction rate of > 90% in the presence of decluttered data and can sustain the same at 60 − 70% even in the presence of noise, such as deletions of peaks and chemical shift deviations. The method tested on NMR data acquired for a mixture of 20 amino acids shows a success rate of 93% in correct recovery of metabolic pathways. The third method developed is a new approach for rapid resonance assignments in proteins based on amino acid selective unlabeling. The method involves choosing a set of multiple amino acid types for selective unlabeling and identifying specific tripeptides surrounding the labeled residues from specific 2D NMR spectra in a combinatorial manner. The methodology directly yields sequence specific resonance assignments, without requiring a contiguously assigned stretch of amino acid residues to be linked, and is applicable to deuterated proteins. The fourth method involves a simple approach to rapidly identify amino acid types in proteins from a 2D NMR spectrum. The method is based on the fact that 13Cβ chemical shifts of different amino acid types fall in distinct spectral regions. By evolving the 13C chemical shifts in the conventional HNCACB or HN(CO)CACB type experiment for a single specified delay period, the phase of the cross peaks of different amino acid residues are modulated depending on their 13Cβ chemical shift values. Following this specified evolution period, the 2D HN projections of these experiments are acquired. The 13C evolution period can be chosen such that all residues belonging to a given set of amino acid types have the same phase pattern (positive or negative) facilitating their identification. This approach does not re-quire the preparation of any additional samples, involves the analysis of 2D [15N,1H] HSQC-type spectra obtained from the routinely used triple resonance experiments with minor modifications, and is applicable to deuterated proteins. Finally, the practical application of these methods for laboratory research is presented. PROMEB and ChemSMP is used to study cancer cell metabolism in previously unexplored oncogenic cell line. PROMEB helped in assigning a differential metabolite present at high concentration in cancer cell line compared to control non-cancerous cell line. ChemSMP revealed active metabolic pathways responsible for regulating energy homeostasis of cancer cells which were previously reported in literature. The two methods developed for rapid protein resonance assignments can be used in applications such as identifying active-site residues involved in ligand binding, phosphorylation, or protein-protein interactions. The phase modulated experiments will be useful for quick assignment of signals that shift during ligand binding or in combination with selective labeling/unlabeling approaches for identification of amino acid types to aid the sequential assignment process. Both the methodology was applied to two proteins: Ubiquitin (8 kDa) and L-IGFBP2 an intrinsically disordered protein (12 kDa), for demonstrating rapid resonance assignment using only set of 2D NMR experiments.