Recent developments in structural proteomics for protein structure determination

The major challenges in structural proteomics include identifying all the proteins on the genome-wide scale, determining their structure-function relationships, and outlining the precise three-dimensional structures of the proteins. Protein structures are typically determined by experimental approaches such as X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. However, the knowledge of three-dimensional space by these techniques is still limited. Thus, computational methods such as comparative and de novo approaches and molecular dynamic simulations are intensively used as alternative tools to predict the three-dimensional structures and dynamic behavior of proteins. This review summarizes recent developments in structural proteomics for protein structure determination; including instrumental methods such as X-ray crystallography and NMR spectroscopy, and computational methods such as comparative and de novo structure prediction and molecular dynamics simulations.     

Combination of NMR spectroscopy and X-ray crystallography offers unique advantages for elucidation of the structural basis of protein complex assembly

NMR spectroscopy and X-ray crystallography are two premium methods for determining the atomic structures of macro-biomolecular complexes. Each method has unique strengths and weaknesses. While the two techniques are highly complementary, they have generally been used separately to address the structure and functions of biomolecular complexes. In this review, we emphasize that the combination of NMR spectroscopy and X-ray crystallography offers unique power for elucidating the structures of complicated protein assemblies. We demonstrate, using several recent examples from our own laboratory, that the exquisite sensitivity of NMR spectroscopy in detecting the conformational properties of individual atoms in proteins and their complexes, without any prior knowledge of conformation, is highly valuable for obtaining the high quality crystals necessary for structure determination by X-ray crystallography. Thus NMR spectroscopy, in addition to answering many unique structural biology questions that can be addressed specifically by that technique, can be exceedingly powerful in modern structural biology when combined with other techniques including X-ray crystallography and cryo-electron microscopy.     

The Mn4Ca photosynthetic water-oxidation catalyst studied by simultaneous X-ray spectroscopy and crystallography using an X-ray free-electron laser

The structure of photosystem II and the catalytic intermediate states of the Mn₄CaO₅ cluster involved in water oxidation have been studied intensively over the past several years. An understanding of the sequential chemistry of light absorption and the mechanism of water oxidation, however, requires a new approach beyond the conventional steady-state crystallography and X-ray spectroscopy at cryogenic temperatures. In this report, we present the preliminary progress using an X-ray free-electron laser to determine simultaneously the light-induced protein dynamics via crystallography and the local chemistry that occurs at the catalytic centre using X-ray spectroscopy under functional conditions at room temperature.     

Time-resolved studies of metalloproteins using X-ray free electron laser radiation at SACLA

BackgroundThe invention of the X-ray free-electron laser (XFEL) has provided unprecedented new opportunities for structural biology. The advantage of XFEL is an intense pulse of X-rays and a very short pulse duration (<10 fs) promising a damage-free and time-resolved crystallography approach.Scope of reviewRecent time-resolved crystallographic analyses in XFEL facility SACLA are reviewed. Specifically, metalloproteins involved in the essential reactions of bioenergy conversion including photosystem II, cytochrome c oxidase and nitric oxide reductase are described.Major conclusionsXFEL with pump-probe techniques successfully visualized the process of the reaction and the dynamics of a protein. Since the active center of metalloproteins is very sensitive to the X-ray radiation, damage-free structures obtained by XFEL are essential to draw mechanistic conclusions. Methods and tools for sample delivery and reaction initiation are key for successful measurement of the time-resolved data.General significanceXFEL is at the center of approaches to gain insight into complex mechanism of structural dynamics and the reactions catalyzed by biological macromolecules. Further development has been carried out to expand the application of time-resolved X-ray crystallography. This article is part of a Special Issue entitled Novel measurement techniques for visualizing ‘live’ protein molecules.     

Opportunities and challenges for time-resolved studies of protein structural dynamics at X-ray free-electron lasers

X-ray free-electron lasers (XFELs) are revolutionary X-ray sources. Their time structure, providing X-ray pulses of a few tens of femtoseconds in duration; and their extreme peak brilliance, delivering approximately 10¹² X-ray photons per pulse and facilitating sub-micrometre focusing, distinguish XFEL sources from synchrotron radiation. In this opinion piece, I argue that these properties of XFEL radiation will facilitate new discoveries in life science. I reason that time-resolved serial femtosecond crystallography and time-resolved wide angle X-ray scattering are promising areas of scientific investigation that will be advanced by XFEL capabilities, allowing new scientific questions to be addressed that are not accessible using established methods at storage ring facilities. These questions include visualizing ultrafast protein structural dynamics on the femtosecond to picosecond time-scale, as well as time-resolved diffraction studies of non-cyclic reactions. I argue that these emerging opportunities will stimulate a renaissance of interest in time-resolved structural biochemistry.     

Protein-lipid interactions in the purple bacterial reaction centre

The purple bacterial reaction centre uses the energy of sunlight to power energy-requiring reactions such as the synthesis of ATP. During the last 20 years, a combination of X-ray crystallography, spectroscopy and mutagenesis has provided a detailed insight into the mechanism of light energy transduction in the bacterial reaction centre. In recent years, structural techniques including X-ray crystallography and neutron scattering have also been used to examine the environment of the reaction centre. This mini-review focuses on recent studies of the surface of the reaction centre, and briefly discusses the importance of the specific protein-lipid interactions that have been resolved for integral membrane proteins.     

Protein-lipid interactions in the purple bacterial reaction centre

The purple bacterial reaction centre uses the energy of sunlight to power energy-requiring reactions such as the synthesis of ATP. During the last 20 years, a combination of X-ray crystallography, spectroscopy and mutagenesis has provided a detailed insight into the mechanism of light energy transduction in the bacterial reaction centre. In recent years, structural techniques including X-ray crystallography and neutron scattering have also been used to examine the environment of the reaction centre. This mini-review focuses on recent studies of the surface of the reaction centre, and briefly discusses the importance of the specific protein-lipid interactions that have been resolved for integral membrane proteins.     

Re-emerging structures: continuing crystallography of the bacterial reaction centre

The reaction centre is nature's solar battery, and is found in a number of variations on a common theme in plants, algae and photosynthetic bacteria. During the last 20 years, a combination of X-ray crystallography, spectroscopy and mutagenesis has provided increasingly detailed insights into the mechanism of light energy transduction in the bacterial reaction centre. This mini-review looks at the application of X-ray crystallography to the bacterial reaction centre, focussing in particular on recent information on the structural consequences of site-directed mutagenesis, the roles played by water molecules in the reaction centre, the mechanism of ubiquinone reduction, and studies of the phospholipid environment of the protein.     

Proteins in action: the physics of structural fluctuations and conformational changes

Structural dynamics is essential for the biological function of proteins. Results from new experimental techniques should be compared with those from previous experiments in order to obtain a consistent picture of the physics of intramolecular fluctuations and conformational changes. The high intensity and time structure of synchrotron radiation have made possible time-resolved X-ray structure analysis and the determination of phonon density spectra through the M?ssbauer effect. By combining results from M?ssbauer absorption spectroscopy, incoherent neutron scattering, low-temperature crystallography and optical spectroscopy, a physical picture of protein dynamics emerges.     

Synthesis and antimicrobial studies of silver N-heterocyclic carbene complexes bearing a methyl benzoate substituent

Due to the properties of silver as an antimicrobial, our research group has synthesized many different silver carbene complexes. Two new silver N-heterocyclic carbene complexes derived from 4,5-dichloroimidazole and theobromine bearing methyl benzoate substituents were synthesized by in situ carbene formation using silver acetate as the base in the reaction. The new compounds were fully characterized by several methods including NMR spectroscopy and X-ray crystallography. Preliminary antimicrobial efficacy studies against Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli were conducted. The results of this study demonstrated antimicrobial efficacy of the two complexes comparable to silver nitrate, showing their potential for use in the treatment of bacterial infections.     

X-ray structure determination of the glycine cleavage system protein H of Mycobacterium tuberculosis using an inverse Compton synchrotron X-ray source

Structural genomics discovery projects require ready access to both X-ray diffraction and NMR spectroscopy which support the collection of experimental data needed to solve large numbers of novel protein structures. The most productive X-ray crystal structure determination laboratories make extensive use of tunable synchrotron X-ray light to solve novel structures by anomalous diffraction methods. This requires that frozen cryo-protected crystals be shipped to large multi acre synchrotron facilities for data collection. In this paper we report on the development and use of the first laboratory-scale synchrotron light source capable of performing many of the state-of-the-art synchrotron applications in X-ray science. This Compact Light Source is a first-in-class device that uses inverse Compton scattering to generate X-rays of sufficient flux, tunable wavelength and beam size to allow high-resolution X-ray diffraction data collection from protein crystals. We report on benchmarking tests of X-ray diffraction data collection with hen egg white lysozyme, and the successful high-resolution X-ray structure determination of the Glycine cleavage system protein H from Mycobacterium tuberculosis using diffraction data collected with the Compact Light Source X-ray beam.     

Dynamic and structural properties of glucose oxidase enzyme

The catalytic oxidation of β-D-glucose by the enzyme glucose oxidase involves a redox change of the flavin coenzyme. The structure and the dynamics of the two extreme glucose oxidase forms were studied by using infrared absorption spectroscopy of the amide I′ band, tryptophan fluorescence quenching and hydrogen isotopic exchange. The conversion of FAD to FADH₂ does not change the amount of α-helix present in the protein outer shell, but reorganises a fraction of random coil to β-sheet structure. The dynamics of the protein interior vary with the redox states of the flavin without affecting the motions of the structural elements near the protein surface. From the structure of glucose oxidase given by X-ray crystallography, these results suggest that the dynamics of the interface between the two monomers are involved in the catalytic mechanism. Received: 27 December 1996 / Accepted: 18 July 1997     

Atomic Resolution Structure of a Protein Prepared by Non-Enzymatic His-Tag Removal. Crystallographic and NMR Study of GmSPI-2 Inhibitor

Purification of suitable quantity of homogenous protein is very often the bottleneck in protein structural studies. Overexpression of a desired gene and attachment of enzymatically cleavable affinity tags to the protein of interest made a breakthrough in this field. Here we describe the structure of Galleria mellonella silk proteinase inhibitor 2 (GmSPI-2) determined both by X-ray diffraction and NMR spectroscopy methods. GmSPI-2 was purified using a new method consisting in non-enzymatic His-tag removal based on a highly specific peptide bond cleavage reaction assisted by Ni(II) ions. The X-ray crystal structure of GmSPI-2 was refined against diffraction data extending to 0.98 Å resolution measured at 100 K using synchrotron radiation. Anisotropic refinement with the removal of stereochemical restraints for the well-ordered parts of the structure converged with R factor of 10.57% and R _free of 12.91%. The 3D structure of GmSPI-2 protein in solution was solved on the basis of 503 distance constraints, 10 hydrogen bonds and 26 torsion angle restraints. It exhibits good geometry and side-chain packing parameters. The models of the protein structure obtained by X-ray diffraction and NMR spectroscopy are very similar to each other and reveal the same β₂αβ fold characteristic for Kazal-family serine proteinase inhibitors.     

Structure and binding properties of hen lysozyme modified at tryptophan 62

The structure of a derivative of hen egg-white lysozyme (EC 3.2.1.17) modified by N-bromosuccinimide at Trp62 has been studied by both ¹H nuclear magnetic resonance spectroscopy and X-ray crystallography. It was shown that this modification, changing the tryptophan residue to an oxindolealanine² residue, only causes minor structural changes at the site of the modification, and that the overall structure of the native enzyme is maintained in the derivative. Both diastereomers of the oxindolealanine-62 lysozyme were observed by the two methods employed, in accordance with previous observations (Norton & Allerhand, 1976). The pK values of the catalytically important carboxyl groups of Glu35 and Asp52 were identical in the native enzyme and its derivative. However, the modified enzyme is virtually inactive in the hydrolysis of the cell-wall mucopolysaccharide of Micrococcus lysodeikticus. The binding of N-acetylglucosamine oligosaccharides to both native lysozyme and Ox-62 lysozyme was studied by nuclear magnetic resonance spectroscopy, observing the perturbations on the lysozyme ¹H n.m.r. resonances, and differences in the perturbations of the two systems demonstrated that binding of (GlcNAc)₃ in particular was not identical in the two systems. The structure of Ox-62 lysozyme-(GlcNAc)₃ was studied by X-ray crystallography and it was shown that only two GlcNAc residues make contact with the enzyme, binding the reducing end residue in a similar mode as the α-anomeric form of GlcNAc binds to the native enzyme (Blake et al., 1967a). On the basis of the results obtained by X-ray crystallography and ¹H n.m.r. spectroscopy, the lack of enzymatic activity of the Ox-62 lysozyme arises from the obstruction by the oxindolealanine residue of sub-site B of the active site, preventing productive binding of the substrate.     

Effect of gem 2,2′-disubstitution and base in the formation of spiro- and ansa-1,3-propandioxy derivatives of cyclotriphosphazenes

The gem-dialkyl effect has been investigated in the reactions of cyclotriphosphazene, N₃P₃Cl₆1, with various 2,2′-derivatives of 1,3-propandiol, CXY(CH₂OH)₂, in either THF or DCM to form spiro (6-membered) and ansa (8-membered ring) derivatives. The reactions were made with a number of symmetrically-substituted (X = Y, methyl, ethyl, n-butyl and a malonate ester) and unsymmetrically-substituted (X ≠ Y, methyl/H, phenyl/H, methyl/n-propyl, ethyl/n-butyl and Br/NO₂) 1,3-propandiols. The products were analysed by ¹H and ³¹P NMR spectroscopy and some of the spiro and ansa derivatives were also characterized by X-ray crystallography. Reactions of 1 with unsymmetrically-substituted 1,3-propandiols results in the formation of two structural isomers of ansa-substituted compounds, both isomers (endo and exo) have been structurally-characterized by X-ray crystallography for the ethyl/n-butyl derivative. It is found that the regioselectivity of the reaction is changed when the base is changed. The relative proportions of spiro and ansa compounds formed under different reaction conditions were quantified by ³¹P NMR measurements of the reaction mixtures. The results were rationalised mainly in terms of the electronic effect of the substituents, whereas the steric effect has a secondary role in the formation of both spiro and ansa compounds.     

Untangling structure–function relationships in the rhomboid family of intramembrane proteases

Rhomboid proteases are a family of integral membrane proteins that have been implicated in critical regulatory roles in a wide array of cellular processes and signaling events. The determination of crystal structures of the prokaryotic rhomboid GlpG from Escherichia coli and Haemophilus influenzae has ushered in an era of unprecedented understanding into molecular aspects of intramembrane proteolysis by this fascinating class of protein. A combination of structural studies by X-ray crystallography, and biophysical and spectroscopic analyses, combined with traditional enzymatic and functional analysis has revealed fundamental aspects of rhomboid structure, substrate recognition and the catalytic mechanism. This review summarizes these remarkable advances by examining evidence for the proposed catalytic mechanism derived from inhibitor co-crystal structures, conflicting models of rhomboid-substrate interaction, and recent work on the structure and function of rhomboid cytosolic domains. In addition to exploring progress on aspects of rhomboid structure, areas for future research and unaddressed questions are emphasized and highlighted. This article is part of a Special Issue entitled: Intramembrane Proteases.     

The role of high-resolution structural studies in the development of commercial enzymes.

Recent developments in both NMR and X-ray crystallography allow the analysis of commercial enzymes in unprecedented detail. The novel methods provide detailed insights into protein dynamics, establish the existence of special catalytic hydrogen bonds and define the ionization states at the enzyme active site. A more detailed understanding of how the changes in structure are related to altered function should facilitate the design of future commercial enzymes with improved performance for different environmental conditions.     

Synchrotron crystallography

The past decade has seen an explosive growth in atomic-level structures determined by X-ray crystallography. Synchrotron radiation and a number of technical advances related quite directly to its development have fueled this growth. With the most recent advances coming to be used collectively and new resources being built, the foundation is laid for a dramatic further expansion of synchrotron crystallography in the next decade. Both the high-throughput applications of structural genomics and also the challenging studies of macromolecular machinery are expected to flourish.