Doctorat en Sciences / info:eu-repo/semantics/nonPublished

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The automated protein structure analysis (APSA) has been developed that describes protein structure via its backbone in a novel way. APSA generates a smooth line for the backbone which is completely described using curvature κ and torsion τ as a function of arc lengths. Diagrams of κ(s) and τ(s) reveal conformational features as typical patterns. In this way ideal and natural helices (α, 310 and π) and β-strands (left and right-handed, parallel and antiparallel) can be rapidly distinguished, their distortions classified, and a detailed picture of secondary structure developed. Such foundations make it possible to qualitatively and quantitatively compare domain structure utilizing calculated κ(s) and τ(s) patterns of proteins. Focusing on the torsion diagrams alone, 16 regions of τ(s) values that correspond to unique groups of conformations have been identified and encoded into 16 letters. The entire protein backbone is described, effectively projecting its three-dimensional (3D) conformation into a one-dimensional (1D) string of letters called the primary code (3D-ID projection), which is APSA's conformational equivalent of a protein's primary structure. The secondary structure is obtained from specific patterns of the primary code (resulting in secondary code). The letter code is used to describe supersecondary structure, which involves a unique characterization of the tum. It contains sufficient information to reconstruct the overall shape of a protein in an unambiguous 1D→3D translation step. Therefore, it is possible to classify supersecondary structure with the help of the letter code in form of a novel labeling system (F.#.M.X.O.L.N.R.U.S) that collects information on the relative orientations of tum and flanking structures (helices, strands). The overall shape of supersecondary structure is obtained by partitioning the surrounding space into octants and cones and assigning the parts of a supersecondary structure to these sub spaces via its labels. This approach can be easily extended to tertiary structure.

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Cryo-electron microscopy (cryo-EM) is a structural biology technique that determines the structure of proteins and macromolecular complexes using the transmission electron microscope under cryogenic conditions. In my Ph.D. studies, I took advantage of this technique, in the study of dynamical features of ribosomes in both eukaryotes and prokaryotes. In Chapter 2, I report my graduate research on the investigation of ribosomes from the human malaria parasite, Plasmodium falciparum, using single-particle cryo-EM. In collaboration with Dr. Jeffrey Dvorin at Harvard Medical School, we obtained five cryo-EM reconstructions of ribosomes purified from P. falciparum blood-stage schizonts, and discovered structural and dynamical features that differentiate the ribosomes of P. falciparum from those of the mammalian system. Moreover, we discovered that RACK1, a necessary ribosomal protein in eukaryotes, does not specifically co-purify with the 80S fraction in the P. falciparum schizonts stage and would mainly function in a ribosome-unbound, free state during the blood-stage. More extensive studies, using cryo-EM methodology, of translation in the parasite, will provide structural knowledge that could help in the design of effective anti-malaria drugs. In Chapter 3, I describe the cryo-EM studies of the Saccharomyces cerevisiae ribosome in response to a carbon source switch. In collaboration with Dr. Andrew Link at Vanderbilt University, we obtained reconstructions of the 80S ribosomes at selected time points after the glucose-to-glycerol carbon source shift, and observed that a fraction of ribosomes lacked densities for r-proteins, mainly eS1 (yeast rpS1) on the 40S subunit and uL16 (yeast rpL10) on the 60S subunit. We found that the binding ratio of eS1 and uL16 to ribosomes changed as a function of time, consistent with the change in translational activities as gauged by polysome profiling. On the basis of these observations, along with previous structural and genetics studies, we propose that rapid control of translation is exerted through the dissociation of r-protein eS1/rpS1 and uL16/rpL10 from the ribosome. Our studies thus open a new venue on the exploration of S. cerevisiae’s rapid adaption to carbon source shifts at the level of translation. In Chapter 4, I have documented a collaborative work on the development and application of a new technique, time-resolved cryo-EM, which can be used to study processes involving two reaction partners on a sub-second time scale. With my colleagues at the Frank and Gonzalez labs at Columbia University, we successfully applied this method to study the process of E. coli ribosomal subunits association. By mixing and reacting the two subunits for 60 ms and 140 ms, we captured the association reaction in a pre-equilibrium state, and detected different conformations of E. coli 70S ribosomes. With the current capability of this mixing-spraying method to visualize multiple states of molecules in a sub-second reaction, we expect to be able to standardize this method and apply it to more challenging biological processes, such as translation recycling and initiation processes.

The structural dynamics of proteins and other macromolecules typically serve crucial roles for their respective biological function. While rigid protein structures are used in classic “lock and key” descriptions of enzymology and receptor-ligand interactions, more and more evidence suggest that the majority of molecular interactions occur on the spectrum between induced-fit binding and conformational selection binding. This model of biomolecular interaction requires, to differing degrees, conformation plasticity and dynamics of the protein itself. To characterize the determinants and implications of protein dynamics, there exists no more suited biophysical technique than nuclear magnetic resonance (NMR) spectroscopy. This method is capable of probing the individual atomic nuclei of proteins in a site-specific manner. Furthermore, NMR spectroscopy is unique in being able to access timescales from picoseconds to seconds, providing information on events from bond vibration and libration to protein folding and ligand binding. The breadth of biophysical information accessible by NMR spectroscopy has led to its widespread use in the study of protein dynamics. The work presented herein involves i) the use of NMR for investigation of structure and dynamics in two separate biological systems that demonstrate a high degree of flexibility for folded proteins and ii) the improvement of pulse sequences and methodology for better characterizing picosecond to nanosecond backbone and side-chain dynamics. The organizing principle of this work, which is best exemplified in the structural studies of the piRNA-pathway protein Gametocyte-specific factor 1, is the unmatched capability of NMR spectroscopy to decipher molecular details within dynamic protein systems. First, the molecular structure and RNA-binding properties of gametocyte-specific factor 1 (GTSF1) of the piRNA effector pathway were investigated. A partially disordered protein with two Zn finger domains, the work presented here describes the isolation of a GTSF1 protein construct amendable to study by NMR spectroscopy. Chemical shift assignment of GTSF1 allowed site-specific observation of amide correlations, which established the basis for NMR structure calculation of GTSF1 and the evaluation of binding to candidate RNA sequences, with goal of the identification of an in vivo RNA binding partner for GTSF1. The work presents compelling data that indicate GTSF1 Zn finger 1 specifically binds a motif GGUUC(G/A) RNA, which in this study was found in the T-arm loop of transfer RNA. Zn finger 2 is affected by the interaction with RNA, but the available structural and binding data indicate that the second Zn finger is a more dynamic, breathable entity, supported by cysteine chemical shift and structural differences between the two GTSF1 Zn fingers. Although it’s currently speculative, the function of GTSF1 might first require binding of RNA to the more stable Zn finger 1, which then leaves Zn finger 2 poised for binding to another molecular species. tRNA-derived fragments that include the T-arm TC loop have been recently implicated in silencing of transposable elements in mammalian cells. GTSF1, which was identified in a genetic screen for piRNA-pathway proteins as vitally required for gene silencing, might plausibly act as a sensor of transcription of transposable elements and help initiate Piwi-piRISCs-mediated chromatin modification and heterochromatin formation. Next, NMR spectroscopy is used to investigate protein thermostability in psychrophilic (cold-loving) cytochrome c552. Isolated from the bacterium Colwellia psychrerythraea (Cp), previous work has implicated two conserved Cpcyt c552 methionine residues, which are both conserved across psychrophilic and psychrotolerant cytochromes, as acting in dynamical ligand substitution with a third methionine that is the axial heme ligand. It is proposed that elevated backbone dynamics in these methionine residues and the ability for them swap into the axial ligand position accounts for an uncharacteristically high melting temperature (Tm) compared to meso- and thermophile c-type cytochromes. Progress was made in NMR sample preparation and backbone chemical shift assignment of both redox states of Cpcyt c552, and insight from 1D 1H NMR experiments focused on the heme group bound to Cp cytochrome c552 is discussed. Additionally, chemical shifts are used to predict protein dynamics as a first test of a multiple methionine axial ligand hypothesis. Initial data analysis predicts relatively large measures of Random Coil Index for residues surrounding the native axial heme ligand, and shows the hyperfine shifts localized to the residues surrounding the heme. Future experiments will selectively record methyl group dynamics of methionine residues for elucidation of rate constants of methionine substitution and to determine the structural properties of this minor conformation. Finally, two NMR methodology studies are presented in this thesis: a novel simultaneous-acquisition TROSY pulse sequence for measurement of backbone spin relaxation rates (R1 and {1H}-15N heteronuclear NOE) and a side-chain 2H spin relaxation method for using multifield experimental datasets for better sampling of the spectral density function. Together, these pulse sequences represent significant advancements in NMR measurement of microscopic rate constants and more nuanced detail of protein dynamics.

The work described in this thesis is concerned with the development and application of real-time photo-CIDNP (Chemically Induced Dynamic Nuclear Polarisation) to the study of protein structure and folding. Chapters 1 and 2 introduce the protein folding problem, and its study by NMR, then go on to elucidate the mechanisms behind the photo-CIDNP phenomenon. Chapter 3 applies photo-CIDNP spectroscopy to the study of a small cytochrome protein. The difficulties of performing these experiments on chromophore-containing proteins are discussed. Chapter 4 begins with the development of a rapid mixing device for use in real-time NMR and CIDNP studies. Experiments used to characterise the device are presented. This chapter then goes on to describe CIDNP pulse labelling experiments, used to investigate the surface structure of some molten globule states of two a-lactalbumins. This chapter concludes with an application of the rapid mixing device to the real-time refolding of hen egg white lysozyme. Chapter 5 extends the work of the previous chapter, studying the real-time refolding of bovine pancreatic ribonuclease A. Refolding studies are performed from different denaturing conditions, and the effects of sample heating during the real-time CIDNP experiment are discussed. Chapter 6 describes the use of illumination during an NMR experiment to study the conformational changes in a plant blue light receptor protein, phototropin. The structural changes are characterised with 2-dimensional NMR spectroscopy and photo-CIDNP. The kinetics of the ground state recovery are also investigated by real-time NMR spectroscopy. Chapter 7 uses calculated hyperfine coupling constants and a radical pair diffusion model from the literature to simulate the nuclear polarisation obtained for the amino acid tryptophan. Comparisons are made between theory and experiment. Chapter 8 describes the structural characterisation of a homologous series of de novo peptides, designed for subsequent use in EPR experiments when derivatised with a suitable spin label.

This thesis describes the use of nuclear magnetic resonance techniques to determine the structures of two proteins in solution, hen egg-white lysozyme and human interleukin-4. Using 2D ¹H methods an extensive set of structural data has been collected for hen lysozyme (1158 NOE distance restraints, 68 o and 24 _?1 dihedral angle restraints) and these data have been used in distance geometry-dynamical simulated annealing calculations to determine an ensemble of NMR structures for the protein. The overall Ca RMSD from the average for a set of 16 calculated structures is 1.8 ± 0.2 A but, excluding 14 residues in exposed disordered regions, this value reduces to 1.3 ± 0.2 Å. Regions of secondary structure, and particularly the four a helices, are well defined (Ca RMSD 0.8 ± 0.3 Å for helices). Detailed comparisons of the NMR structures with crystal structures of the protein have shown the close similarity of the main chain fold and the conformation of interior side chains in solution and in crystals. ³J_aß coupling constant measurement have indicated, however, that the conformational mobility of the side chains of many surface residues is significantly more pronounced than an individual crystal structure would suggest. For human interleukin-4, a strategy involving ¹⁵N and ¹³C labelled recombinant protein together with heteronuclear 3D NMR techniques has been employed to determine the structure of the protein. The work has provided the first structure for this protein, a left-handed four helix bundle with an up-up-down-down connectivity. For an ensemble of 10 final calculated NMR structures there is a Ca RMSD from the average of 1.6 ± 0.4 Å, the definition of the helical core of the protein being particularly good (0.8 ± 0.2 Å). There is, however, some disorder in the long overhand loops of the structure; this reflects the unusually high conformational mobility of these regions. Comparison of the interleukin-4 structure with proteins with related folds, particularly members of the haemopoietic cytokine superfamily, suggests that the fold found here for interleukin-4 may be the adopted structure throughout this cytokine superfamily. In a postscript to this thesis the NMR structure of human interleukin-4 is shown to have a very similar conformation to a crystal structure of the protein which has been solved very recently.

Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2008. / Committee Chair: Skolnick, Jeffrey; Committee Member: Fernandez, Facundo; Committee Member: Jordan, King; Committee Member: McDonald, John; Committee Member: Sherrill, David. Part of the SMARTech Electronic Thesis and Dissertation Collection.

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