This thesis describes the structural studies of MPR-TM (residues 649-705) of HIV-1 gp41 by X-ray crystallography. MPR-TM was fused with different fusion proteins to improve the membrane protein overexpression. The expression level of MPR-TM was improved by fusion to the C-terminus of the Mistic protein, yielding ∼1 mg of pure MPR-TM protein per liter cell culture. The fusion partner Mistic was removed for final crystallization. The isolated MPR-TM protein was biophysically characterized and is a monodisperse candidate for crystallization. However, no crystal with diffraction quality was obtained even after extensive crystallization screens. A novel construct was designed to overexpress MPR-TM as a maltose binding protein (MBP) fusion. About 60 mg of MBP/MPR-TM recombinant protein was obtained from 1 liter of cell culture. Crystals of MBP/MPR-TM recombinant protein could not be obtained when MBP and MPR-TM were separated by a 42 amino acid (aa)-long linker but were obtained after changing the linker to three alanine residues. The crystals diffracted to 2.5 Å after crystallization optimization. Further analysis of the diffraction data indicated that the crystals are twinned. The final structure demonstrated that MBP crystallized as a dimer of trimers, but the electron density did not extend beyond the linker region. We determined by SDS-PAGE and MALDI-TOF MS that the crystals contained MBP only. The MPR-TM of gp41 might be cleaved during or after the process of crystallization. Comparison of the MBP trimer reported here with published trimeric MBP fusion structures indicated that MBP might form such a trimeric conformation under the effect of MPR-TM.
The first part of this dissertation describes the optimization of the previously established protocol for the H. modesticaldum reaction center isolation. Subsequently, electron transfer is characterized by ultrafast spectroscopy; the primary electron acceptor, a chlorophyll a derivative, is reduced in ~25 ps, and forward electron transfer occurs directly to a 4Fe-4S cluster in ~650 ps without the requirement for a quinone intermediate. A 2.2-angstrom resolution X-ray crystal structure of the homodimeric heliobacterial reaction center is solved, which is the first ever homodimeric reaction center structure to be solved, and is discussed as it pertains to the structure-function relationship in energy and electron transfer. The structure has a transmembrane helix arrangement similar to that of Photosystem I, but differences in antenna and electron transfer cofactor positions explain variations in biophysical comparisons. The structure is then compared with other reaction centers to infer evolutionary hypotheses suggesting that the ancestor to all modern reaction centers could reduce mobile quinones, and that Photosystem I added lower energy cofactors to its electron transfer chain to avoid the formation of singlet oxygen.
In the second part of this dissertation, hydrogen production rates of H. modesticaldum are quantified in multiple conditions. Hydrogen production only occurs in cells grown without ammonia, and is further increased by removal of N2. These results are used to propose a scheme that summarizes the hydrogen-production metabolism of H. modesticaldum, in which electrons from pyruvate oxidation are shuttled through an electron transport pathway including the reaction center, ultimately reducing nitrogenase. In conjunction, electron microscopy images of H. modesticaldum are shown, which confirm that extended membrane systems are not exhibited by heliobacteria.
The availability of X-ray free electron lasers presents an opportunity to study micron-sized crystals that could be triggered (using light, small molecules or physical conditions) to capture macromolecules in action. This method of ‘Time-resolved serial crystallography’ answers key biological questions by capturing snapshots of conformational changes associated with multi-step reactions. This dissertation describes approaches for studying structures of large membrane protein complexes. Both macro and micro-seeding techniques have been implemented for improving crystal quality and obtaining high-resolution structures. Well-diffracting 15-20 micron crystals of active Photosystem II were used to perform time-resolved studies with fixed-target Roadrunner sample delivery system. By employing continuous diffraction obtained up to 2 A, significant progress can be made towards understanding the process of water oxidation.
Structure of Photosystem I was solved to 2.3 A by X-ray crystallography and to medium resolution of 4.8 A using Cryogenic electron microscopy. Using complimentary techniques to study macromolecules provides an insight into differences among methods in structural biology. This helps in overcoming limitations of one specific technique and contributes in greater knowledge of the molecule under study.
ano-crystals have been established for the different variants of the fusion protein. Diffraction patterns were collected by using both conventional and serial femto-second crystallography techniques. The two crystallography techniques showed very interesting differences in both the crystal packing and unit cell dimensions of the same CTB-MPR construct. Although information has been gathered on CTB-MPR, the intact structure of fusion protein was not solved as the MPR region showed only weak electron density or was cleaved during crystallization of macroscopic crystals. The MPR region is present in micro
ano-crystals, but due to the severe limitation of the Free Electron Laser beamtime, only a partial data set was obtained and is insufficient for structure determination. However, the work of this thesis has established methods to purify large quantities of CTB-MPR and has established procedures to grow crystals for X-ray structure analysis. This has set the foundation for future structure determination experiments as well as immunization studies.
Most serial crystallography experiments have relied on delivering sample in the mother liquor focused into a stream by compressed gas. This liquid stream moves at a fast rate, meaning that most of the valuable sample is wasted. For this reason, the liquid jet can require 10-100 milligrams of sample for a complete data set. Agarose has been developed as a slow moving microcrystal carrier to decrease sample consumption and waste. The agarose jet provides low background, no Debye-Sherrer rings, is compatible for sample delivery in vacuum environments, and is compatible with a wide variety of crystal systems. Additionally, poly(ethylene oxide) which is amenable for data collection in atmosphere has been developed for synchrotron experiments. Thus this work allows sample limited proteins of difficult to crystallize systems to be investigated by serial crystallography.
Time-resolved serial X-ray crystallography (TR-SX) studies have only been employed to study light-triggered reactions in photoactive systems. While these systems are very important, most proteins in Nature are not light-driven. However, fast mixing of two liquids, such as those containing enzyme protein crystals and substrates, immediately before being exposed to an X-ray beam would allow conformational changes and /or intermediates to be seen by diffraction. As a model, 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS), has been developed for TR-SX. This enzyme initializes the first step of lipopolysaccharide synthesis by a net aldol condensation between arabinose-5-phosphate, phosphoenol pyruvate, and water. During this reaction, a short lived intermediate is formed and has been observed on a millisecond timescale using other methods. Thus KDO8PS is an ideal model protein for studying diffusion times into a crystal and short mixing times (<10 ms). For these experiments, microcrystals diffracting to high resolution have been developed and characterized.
Rosalind Elsie Franklin worked with X-ray crystallography at King's College London, UK, and she helped determine the helical structure of DNA in the early 1950s. Franklin's research helped establish molecular genetics, a field that investigates how heredity works on the molecular level. The discovery of the structure of DNA also made future research possible into the molecular basis of embryonic development, genetic disorders, and gene manipulation.
In February 1953, Linus Pauling and Robert Brainard Corey, two scientists working at the California Institute of Technology in Pasadena, California, proposed a structure for deoxyribonucleic acid, or DNA, in their article “A Proposed Structure for the Nucleic Acids,” henceforth “Nucleic Acids.” In the article, Pauling and Corey suggest a model for nucleic acids, including DNA, that consisted of three nucleic acid strands wound together in a triple helix. “Nucleic Acids” was published in Proceedings of the National Academy of Sciences shortly after scientists came to the consensus that genes, the biological factors that control how organisms develop, contained DNA. Though scientists proved Pauling and Corey’s model incorrect, “Nucleic Acids” helped scientists understand DNA’s structure and function as genetic material.