Intrinsic Neutral and Anionic Structures of Glutathione

Gas‐phase intrinsic structures of intact neutral and anionic glutathione (GSH) have been determined by means of a combination of negative ion photo‐electron spectroscopy and quantum chemistry calculations. The inferred structures of the neutral parents of those peptide anions are canonical (non‐zwitterionic). These intrinsic structures are compared to those already known in aqueous solution or determined by crystallography in binding sites of enzymes.     

Exchange in vitro of Subunits between Enzymes from Different Organisms: Chimeras of Enzymes

Most intracellular enzymes are made up of several identical or different subunits. The more remote any two organisms are phylogenetically, the greater will be the differences in amino acid sequence of a given enzyme. Nevertheless, numerous examples exist of specific association between chemically different subunits, or even of the formation of enzyme chimeras. They span not only the boundaries between related organisms but also the deep rift between prokaryotic and eukaryotic cells. Exchange of subunits between enzymes of similar activity but differing origin can be rationalized by assuming enzymes to posses functionally defined types of three-dimensional structures.     

SOS-NMR: a saturation transfer NMR-based method for determining the structures of protein-ligand complexes

An NMR-based alternative to traditional X-ray crystallography and NMR methods for structure-based drug design is described that enables the structure determination of ligands complexed to virtually any biomolecular target regardless of size, composition, or oligomeric state. The method utilizes saturation transfer difference (STD) NMR spectroscopy performed on a ligand complexed to a series of target samples that have been deuterated everywhere except for specific amino acid types. In this way, the amino acid composition of the ligand-binding site can be defined, and, given the three-dimensional structure of the protein target, the three-dimensional structure of the protein-ligand complex can be determined. Unlike earlier NMR methods for solving the structures of protein-ligand complexes, no protein resonance assignments are necessary. Thus, the approach has broad potential applications--especially in cases where X-ray crystallography and traditional NMR methods have failed to produce structural data. The method is called SOS-NMR for structural information using Overhauser effects and selective labeling and is validated on two protein-ligand complexes: FKBP complexed to 2-(3'-pyridyl)-benzimidazole and MurA complexed to uridine diphosphate N-acetylglucosamine.     

Travelling wave ion mobility mass spectrometry studies of protein structure: biological significance and comparison with X-ray crystallography and nuclear magnetic resonance spectroscopy measurements

The three-dimensional conformation of a protein is central to its biological function. The characterisation of aspects of three-dimensional protein structure by mass spectrometry is an area of much interest as the gas-phase conformation, in many instances, can be related to that of the solution phase. Travelling wave ion mobility mass spectrometry (TWIMS) was used to investigate the biological significance of gas-phase protein structure. Protein standards were analysed by TWIMS under denaturing and near-physiological solvent conditions and cross-sections estimated for the charge states observed. Estimates of collision cross-sections were obtained with reference to known standards with published cross-sections. Estimated cross-sections were compared with values from published X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy structures. The cross-section measured by ion mobility mass spectrometry varies with charge state, allowing the unfolding transition of proteins in the gas phase to be studied. Cross-sections estimated experimentally for proteins studied, for charge states most indicative of native structure, are in good agreement with measurements calculated from published X-ray and NMR structures. The relative stability of gas-phase structures has been investigated, for the proteins studied, based on their change in cross-section with increase in charge. These results illustrate that the TWIMS approach can provide data on three-dimensional protein structures of biological relevance.     

N-acylamidine palladium(II) complexes. Synthesis,structures, and catalytic activity for Suzuki-Miyaura cross-coupling reactions: experiments and DFT calculations

N-Acylamidines 2 are easily prepared by acylation of amidines 1. Upon treatment with PdCl(2)(PhCN)(2), they form 2:1 PdCl(2) complexes 3 with trans configuration, acting as monodentate ligands via the nitrogen atom remote from the carbonyl group. The structures of the complexes 3a-c in the solid state were obtained by X-ray crystallography. As studied by DFT calculations on 2:1 model complexes, many isomeric structures 6 with different conformations and configurations are associated with local minima on the energy hyperface within an energy range of 4 kcal/mol. The complexes 3 show very high catalytic activity in Suzuki-Miyaura cross-coupling reactions, either as isolated crystals or prepared in situ without isolation.     

Electron Diffraction with the Transmission Electron Microscope as a Phase-Determining Diffractometer—From Spatial Frequency Filtering to the Three-Dimensional Structure Analysis of Ribosomes

Chemists recognize X-ray crystal structure analysis and electron microscopy as powerful methods of analysis. In the last 20 years the basic ideas of X-ray diffraction analysis have been extended to the field of electron microscopy, whereby an image-forming apparatus is converted into an electron diffractometer, and through which an old dream of crystallographers can be realized—the measurement of the phase shift of scattered waves, a prerequisite for the direct calculation of structures. Its most important area of application, like that of the X-ray diffractometer, is in three-dimensional structure analysis—in all fields of science. However, beyond crystallography, aperiodic structures (comparable to crystals with a single unit cell) can also be analyzed three-dimensionally. In this progress report, the development of the first idea (spatial frequency filtering) to the analysis of ribosomal particles is outlined. Attention will be focused primarily on quantitative methods for the measurement of scattered rays, which are also usable beyond the conventional limit of resolution, down to atomic resolution. In the course of this work in 1968, the principle of the three-dimensional analysis of native biological crystal structures using the electron microscope, as worked with today in many laboratories, was developed. In Munich, however, further research focused on the three-dimensional analysis of aperiodic and individual (especially biological) objects. The analysis of 50S-subunits of the procaryotic ribosome of E. coli showed surprisingly good reproducibility of the results (although only within the same orientation), allowing the deduction of almost ideal average model structures from a limited number of particles.     

Protective effect of sorbitol on enzymes exposed to microsecond pulsed electric field

Lysozyme (alpha-helix dominant structure) and pepsin (beta-sheet dominant structure) were exposed to microsecond pulsed electric field (PEF) at 3.5x10(6) V/m. The response of enzymes to the stress of PEF was investigated in this study. Unfolding of enzyme structures and disruption of secondary and three-dimensional structures occurred when the exposed PEF dosage exceeds a critical value, which caused the decrease in activity. In this work, sorbitol was found to be effective to stabilize the conformations and activities of enzymes against electric field. The protective effect increased with the increase of concentration of sorbitol.     

Protein structure determination by high-resolution solid-state NMR spectroscopy: application to microcrystalline ubiquitin

High-resolution solid-state NMR spectroscopy has become a promising method for the determination of three-dimensional protein structures for systems which are difficult to crystallize or exhibit low solubility. Here we describe the structure determination of microcrystalline ubiquitin using 2D (13)C-(13)C correlation spectroscopy under magic angle spinning conditions. High-resolution (13)C spectra have been acquired from hydrated microcrystals of site-directed (13)C-enriched ubiquitin. Inter-residue carbon-carbon distance constraints defining the global protein structure have been evaluated from 'dipolar-assisted rotational resonance' experiments recorded at various mixing times. Additional constraints on the backbone torsion angles have been derived from chemical shift analysis. Using both distance and dihedral angle constraints, the structure of microcrystalline ubiquitin has been refined to a root-mean-square deviation of about 1 A. The structure determination strategies for solid samples described herein are likely to be generally applicable to many proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy.     

环糊精及其衍生物的超分子晶体结构研究进展

本文对近年来有关环糊精、环糊精衍生物以及它们与各类客体组装成的超分子包合物的晶体结构研究进行的简要概述。     

Recent advances in protein NMR spectroscopy and their implications in protein therapeutics research

Nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography are the two main methods for protein three-dimensional structure determination at atomic resolution. According to the protein structures deposited in the Protein Data Bank, X-ray crystallography has become the dominant method for structure determination, particularly for large proteins and complexes. However, with the developments of isotope labeling, increase of magnetic field strength, common use of a cryogenic probe, and ingenious pulse sequence design, the applications of NMR spectroscopy have expanded in biological research, especially in characterizing protein dynamics, sparsely populated transient structures, weak protein interactions, and proteins in living cells at atomic resolution, which is difficult if not impossible by other biophysical methods. Although great advances have been made in protein NMR spectroscopy, its applications in protein therapeutics, which represents the fastest growing segment of the pharmaceutical industry, are still limited. Here we review the recent advances in the use of NMR spectroscopy in studies of large proteins or complexes, posttranslation modifications, weak interactions, and aggregation, and in-cell NMR spectroscopy. The potential applications of NMR spectroscopy in protein therapeutic assays are discussed.     

Metalloprotein and metallo-DNA/RNAzyme design: current approaches, success measures, and future challenges

Specific metal-binding sites have been found in not only proteins but also DNA and RNA molecules. Together these metalloenzymes consist of a major portion of the enzyme family and can catalyze some of the most difficult biological reactions. Designing these metalloenzymes can be both challenging and rewarding because it can provide deeper insights into the structure and function of proteins and cheaper and more stable alternatives for biochemical and biotechnological applications. Toward this goal, both rational and combinatorial approaches have been used. The rational approach is good for designing metalloenzymes that are well characterized, such as heme proteins, while the combinatorial approach is better at designing those whose structures are poorly understood, such as metallo-DNA/RNAzymes. Among the rational approaches, de novo design is at its best when metal-binding sites reside in a scaffold whose structure has been designed de novo (e.g., alpha-helical bundles). Otherwise, design using native scaffolds can be equally effective, allowing more choices of scaffolds whose structural stability is often more resistant to multiple mutations. In addition, computational and empirical designs have both enjoyed successes. Because of the limitation in defining structural parameters for metal-binding sites, a computational approach is restricted to mostly metal-binding sites that are well defined, such as mono- or homonuclear centers. An empirical approach, even though it is less restrictive in the metal-binding sites to be designed, depends heavily on one's knowledge and choice of templates and targets. An emerging approach is a combination of both computational and empirical approaches. The success of these approaches can be measured not only by three-dimensional structural comparison between the designed and target enzymes but also by the total amount of insight obtained from the design process and studies of the designed enzymes. One of the biggest advantages of designed metalloenzymes is the potential of placing two different metal-binding sites in the same protein framework for comparison. A final measure of success is how one can utilize the insight gained from the intellectual exercise to design new metalloenzymes, including those with unprecedented structures and functions. Future challenges include designing more complex metalloenzymes such as heteronuclear metal centers with strong nanomolar or better affinities. A key to meeting this challenge is to focus on the design of not only primary but also secondary coordination spheres using a combination of improved computer programs, experimental design, and high-resolution crystallography.     

On the structure of quasi-crystals in the higher-dimensional space

Categories of the generalized crystallography, where structures are tiled into a large number of identical cells, include quasi-identity and quasi-equivalence. The hierarchy of organization levels including the n-dimensional space is considered. In the theory of proportions, irrational numbers, such as e, π, τ, etc., play an important role. The golden ratio τ is basic to the geometry of structures with five- or tenfold symmetry that are called quasi-crystals. The irrational nature of these numbers probably means that in the Euclidean space E3, we deal with projections of the fundamental polyhedra from a higher-dimensional space. Thus, the structures of quasi-crystals in the three-dimensional space represent slices of much more complex assemblies in the higher-dimensional space.     

Similar biological activities of two isostructural ruthenium and osmium complexes

In this study, we probe and verify the concept of designing unreactive bioactive metal complexes, in which the metal possesses a purely structural function, by investigating the consequences of replacing ruthenium in a bioactive half-sandwich kinase inhibitor scaffold by its heavier congener osmium. The two isostructural complexes are compared with respect to their anticancer properties in 1205 Lu melanoma cells, activation of the Wnt signaling pathway, IC(50) values against the protein kinases GSK-3beta and Pim-1, and binding modes to the protein kinase Pim-1 by protein crystallography. It was found that the two congeners display almost indistinguishable biological activities, which can be explained by their nearly identical three-dimensional structures and their identical mode of action as protein kinase inhibitors. This is a unique example in which the replacement of a metal in an anticancer scaffold by its heavier homologue does not alter its biological activity.     

Evolutionary relationship and application of a superfamily of cyclic amidohydrolase enzymes

Cyclic amidohydrolases belong to a superfamily of enzymes that catalyze the hydrolysis of cyclic C-N bonds. They are commonly found in nucleotide metabolism of purine and pyrimidine. These enzymes share similar catalytic mechanisms and show considerable structural homologies, suggesting that they might have evolved from a common ancestral protein. Homology searches based on common mechanistic properties and three-dimensional protein structures provide clues to the evolutionary relationships of these enzymes. Among the superfamily of enzymes, hydantoinase has been highlighted by its potential for biotechnological applications in the production of unnatural amino acids. The enzymatic process for the production of optically pure amino acids consists of three enzyme steps: hydantoin racemase, hydantoinase, and N-carbamoylase. For efficient industrial application, some critical catalytic properties such as thermostability, catalytic activity, enantioselectivity, and substrate specificity require further improvement. To this end, isolation of new enzymes with desirable properties from natural sources and the optimization of enzymatic processes were attempted. A combination of directed evolution techniques and rational design approaches has made brilliant progress in the redesign of industrially important catalytic enzymes; this approach is likely to be widely applied to the creation of designer enzymes with desirable catalytic properties.     

A Structural Basis of Light Energy and Electron Transfer in Biology (Nobel Lecture)

Aspects of intramolecular light energy and electron transfer are discussed for three protein cofactor complexes whose three-dimensional structures have been elucidated by X-ray crystallography: the light harvesting phycobilisomes of cyanobacteria, the reaction center of purple bacteria, and the blue multi-copper oxidases. A wealth of functional data is available for these systems which allows specific correlations to be made between structure and function and general conclusions to be drawn about light energy and electron transfer in biological materials.     

Room-temperature crystallography using a microfluidic protein crystal array device and its application to protein–ligand complex structure analysis

Room-temperature (RT) protein crystallography provides significant information to elucidate protein function under physiological conditions. In particular, contrary to typical binding assays, X-ray crystal structure analysis of a protein–ligand complex can determine the three-dimensional (3D) configuration of its binding site. This allows the development of effective drugs by structure-based and fragment-based (FBDD) drug design. However, RT crystallography and RT crystallography-based protein–ligand complex analyses require the preparation and measurement of numerous crystals to avoid the X-ray radiation damage. Thus, for the application of RT crystallography to protein–ligand complex analysis, the simultaneous preparation of protein–ligand complex crystals and sequential X-ray diffraction measurement remain challenging. Here, we report an RT crystallography technique using a microfluidic protein crystal array device for protein–ligand complex structure analysis. We demonstrate the microfluidic sorting of protein crystals into microwells without any complicated procedures and apparatus, whereby the sorted protein crystals are fixed into microwells and sequentially measured to collect X-ray diffraction data. This is followed by automatic data processing to calculate the 3D protein structure. The microfluidic device allows the high-throughput preparation of the protein–ligand complex solely by the replacement of the microchannel content with the required ligand solution. We determined eight trypsin–ligand complex structures for the proof of concept experiment and found differences in the ligand coordination of the corresponding RT and conventional cryogenic structures. This methodology can be applied to easily obtain more natural structures. Moreover, drug development by FBDD could be more effective using the proposed methodology.

Room temperature protein crystallography and its application to protein–ligand complex structure analysis was demonstrated using a microfluidic protein crystal array device.     

X-ray and NMR crystallography in an enzyme active site: the indoline quinonoid intermediate in tryptophan synthase

Chemical-level details such as protonation and hybridization state are critical for understanding enzyme mechanism and function. Even at high resolution, these details are difficult to determine by X-ray crystallography alone. The chemical shift in NMR spectroscopy, however, is an extremely sensitive probe of the chemical environment, making solid-state NMR spectroscopy and X-ray crystallography a powerful combination for defining chemically detailed three-dimensional structures. Here we adopted this combined approach to determine the chemically rich crystal structure of the indoline quinonoid intermediate in the pyridoxal-5'-phosphate-dependent enzyme tryptophan synthase under conditions of active catalysis. Models of the active site were developed using a synergistic approach in which the structure of this reactive substrate analogue was optimized using ab initio computational chemistry in the presence of side-chain residues fixed at their crystallographically determined coordinates. Various models of charge and protonation state for the substrate and nearby catalytic residues could be uniquely distinguished by their calculated effects on the chemical shifts measured at specifically (13)C- and (15)N-labeled positions on the substrate. Our model suggests the importance of an equilibrium between tautomeric forms of the substrate, with the protonation state of the major isomer directing the next catalytic step.     

Acetylpinnasterol and pinnasterol,ecdysone-like metabolites,from the marine red alga laurenciapinnata yamada

The structures of two sterols, isolated from the title alga and designated as acetylpinnasterol and pinnasterol, were determined on the basis of the X-ray crystallography. These metabolites are the first marine phytosterols with ecdysone-like structures and biological activity as moulting hormones.     

Query generation to search for inhibitors of enzymatic reactions

A method for the generation of intermediates of enzyme-catalyzed reactions is presented. These intermediates can be used as three-dimensional structural queries for searching for inhibitors of enzymatic reactions. The intermediates can be considered as being structurally quite close to transition-state analogues. For this application, a database containing detailed chemical information on metabolic reactions is used. The likely three-dimensional structure of the intermediates of enzyme-catalyzed reactions can be generated from the information in the database. For three reactions catalyzed by the enzymes AMP deaminase (EC code 3.5.4.6), triose phosphate isomerase (EC code 5.3.1.1), and arginase II (EC code 3.5.3.1), we show how a 3D model of these intermediates can be superimposed onto known inhibitors of these enzymes by a program that uses a genetic algorithm. For this, we test different methods for the superimposition using information on the enzymatic binding site, using information on physicochemical properties calculated from the molecular structure, or without having any information in the superimposition process. We show that these inhibitors are most similar to the corresponding intermediates regarding the 3D structure.     

Determination of the stereochemistry of anhydroerythromycin A, the principal degradation product of the antibiotic erythromycin A

Anhydroerythromycin A arises from the acid-catalysed degradation of erythromycin A both in vitro and in vivo. It has negligible antibacterial activity, but inhibits drug oxidation in the liver, and is responsible for unwanted drug-drug interactions. Its structure has 18 chiral centres common with erythromycin A, but C-9 (the spiro carbon) is also chiral in anhydroerythromycin and its stereochemistry has not previously been reported; both 9R- and 9S-anhydroerythromycin A are plausible structures. An understanding of the chirality at C-9 was expected to throw light on the mechanism of acid-catalysed degradation of erythromycin A, a subject that has been debated in the literature over several decades.We now report a determination of the three-dimensional structure of anhydroerythromycin A, including the stereochemistry at C-9, by NMR and molecular modelling. In parallel, the relative stereochemistry of anhydroerythromycin A 2'-acetate was determined by X-ray crystallography. Both compounds were shown to have 9R stereochemistry, and anhydroerythromycin A exhibited considerable conformational flexibility in solution.