X-Ray Diffraction as a Tool for Studying Collagen Structure
Marcel E. Nimni in Collagen, 1988
Ideally, for X-ray crystallography, one is interpreting the X-ray diffraction pattern from a single crystal of a well-characterized component. This contrasts markedly with examining a whole native tissue, which always contains a mixture of molecules. However, X-ray diffraction does not “see” all molecules equally. The components which are well ordered and well oriented will diffract strongly, while unordered molecules will only contribute to the background scattering. In rat tail tendon, collagen constituents over 90% of the dry tissue weight, and the ordered collagen fibrils produce the diffraction pattern. In skin and notochord, the amounts of noncollagenous components become more significant, but the diffraction pattern is dominated by the ordered fibrils, while the unordered proteoglycans or noncollagenous proteins will increase the level of background scattering. If there are noncollagenous components periodically bound to the collagen fibrils, changes in the intensities of reflections are expected. This has been the basis for experiments investigating proteoglycan binding along collagen fibrils5 and for localization of mineral in bone fibrils.6
Dopamine Receptors, Signaling Pathways, and Drugs
Nira Ben-Jonathan in Dopamine, 2020
The determination of the exact structure of the ligand-binding pocket within the DARs has long been a challenging endeavor. Scientists typically resolve the chemical structure of a protein by means of X-ray crystallography. For that, various methods are used to induce the protein to condense into a tightly packed crystal lattice. Once this is achieved, X-rays are delivered to the crystal and the structure of the protein is resolved from the diffraction patterns. However, getting the DAR proteins to crystalize with a bound ligand had been problematic for many years. This difficulty stems from the fact that membrane-embedded receptors are notoriously difficult proteins for crystallization because of their low abundance and hydrophobicity. In fact, of the five DAR subtypes, only the D3R has been successfully crystallized.
3D Particles
Paul Pumpens in Single-Stranded RNA Phages, 2020
The current chapter is simply the logical continuation of Chapter 9. The long gap between these two chapters was necessary to present first two general subjects, namely, the expression of the RNA phage genes and the generation of chimeric VLPs; the knowledge of both is necessary to understand this chapter. Thus, this chapter appears after the genetic engineering of the RNA phages, which was explained in Chapter 19 and the general outline of the chimeric VLPs, which was presented in Chapter 20. As described in the Symmetry section of Chapter 9, the general idea of the spatial structure of the icosahedral RNA phages was clarified in the mid-1970s. The study of high-resolution structures began in the early 1990s with x-ray crystallography and is continued up to now. The first crystals were obtained for the phage preparations, while further investigations quite often preferred the VLPs that were obtained after expression of the RNA phage coat gene in E. coli or yeast. Moreover, since the active studies of the chimeric VLPs began in the mid-1990s, the determination of the three-dimensional (3D) structure was tightly connected with the urgent problem of how to display foreign peptides on the particle surface. To learn the global history of viral architecture, a review article written by the famous crystallographer Michael G. Rossmann (2013) is recommended.
Enhancement of levodopa stability when complexed with β-cyclodextrin in transdermal patches
Published in Pharmaceutical Development and Technology, 2018
Rana Obaidat, Nizar Al-Shar'i, Bassam Tashtoush, Tamara Athamneh
X-ray crystallography is a tool used for identifying the atomic and molecular structure of a crystal. X-ray diffraction of levodopa (Figure 4) confirms the presence of the drug as a crystal6. Major peaks were observed for the crystalline form of levodopa at the following 2θ values: 6.6°, 13.1°, 18.3°,19.7°, 21.3°, 22.7°, 24.9°, 25.9°, 28.6°. It can be surmised from Figure 4 that the characteristic sharp peaks of levodopa disappeared in the levodopa/carbopol, levodopa:βCD/carbopol and levodopa:βCD/xanthan patches confirming the transformation of the drug into its amorphous form. However, the levodopa/xanthan patch showed the presence of characteristic peaks for levodopa at the following 2θ values: 6.6°, 13.1°, 18.3°, 19.7° and 22.7°, which indicate that the drug still appears in its crystalline form in this formulation.
Characterization of the phosphotransacetylase-acetate kinase pathway for ATP production in Porphyromonas gingivalis
Published in Journal of Oral Microbiology, 2019
Yasuo Yoshida, Mitsunari Sato, Takamasa Nonaka, Yoshiaki Hasegawa, Yuichiro Kezuka
Since P. gingivalis produces a large amount of short chain fatty acids, including acetate, propionate, and butyrate [23], it is postulated that the Pta-Ack pathway, which is associated with acetate production, is conserved in P. gingivalis [8,9] (Figure 1). Despite the importance of Pta and Ack in carbon cycling and energy metabolism in P. gingivalis, these enzymes have not been characterized in this microorganism, even though the coding genes have been assigned based on sequence homology [24,25]. In the current study, recombinant Pta (PgPta) and Ack (PgAck) from P. gingivalis ATCC 33277, encoded by pta (PGN_1179) and ack (PGN_1178), respectively, were prepared and enzymatically characterized. In addition, crystal structures were determined by X-ray crystallography. Based on the structures, several putative functional residues were identified and their contribution to catalytic activity was evaluated by site-directed mutagenesis. Furthermore, we examined the essentiality of pta and ack in P. gingivalis.
Fragment-based screening with natural products for novel anti-parasitic disease drug discovery
Published in Expert Opinion on Drug Discovery, 2019
Crystallographic fragment screening is one of the major techniques for FBDD in which protein crystals are soaked with high concentrations of fragments. Specific binding of ligands to the protein is detected from analysis of X-ray diffraction data collected from crystals. The main advantage of X-ray crystallography is it can provide an immediate model of the fragment binding to the protein, however, the main disadvantage is that it requires a suitable crystal system for compounds bound to target proteins that can be a major issue for some targets [40]. Crystallographic fragment screening was used to find inhibitors of Trypanosoma brucei nucleoside 2-deoxyribosyltransferase (TbNDRT) [41]. Thirty-one fragment cocktails were generated from 304 commercially available fragments by mixing 7–10 compounds into one cocktail based on chemical properties. Co-crystallographic screening of the 31 cocktails with TbNDRT resulted in 69 crystals, in which four ligands (49–52) (Figure 12) were identified in the active site. The four ligands were screened against bloodstream form T. brucei cell cultures, all showing growth inhibition effects with ED50 between 0.12 and 1.34 mM.
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