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BBA - Proteins and Proteomics (v.1814, #6)
X-ray crystallography marries spectroscopy to unveil structure and function of biological macromolecules
by A.R. Pearson; Andrea Mozzarelli (pp. 731-733).
X-ray crystallography marries spectroscopy to unveil structure and function of biological macromolecules
by A.R. Pearson; Andrea Mozzarelli (pp. 731-733).
Protein crystal microspectrophotometry by Luca Ronda; Stefano Bruno; Stefano Bettati; Andrea Mozzarelli (pp. 734-741).
Single crystal microspectrophotometry has emerged as a valuable technique for monitoring molecular events that take place within protein crystals, thus tightly coupling structure to function. Absorption and fluorescence spectra, ligand binding affinities and kinetic constants can be determined, allowing i) the definition of the experimental conditions for X-ray crystallography experiments and their interpretation, ii) the assessment of whether crystal lattice forces have altered conformational equilibria, iii) the comparison with data obtained in solution. Microspectrophotometric measurements using oriented crystals and linearly polarized light are carried out usually off-line with respect to X-ray data collection and are aimed at an in- depth characterization of protein function in the crystal, leading to robust structure–function relationships. The power of this approach is highlighted by reporting a few case studies, including hemoglobins, pyridoxal 5′-phosphate-dependent enzymes and acetylcholinesterases. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.
Keywords: Abbreviations; GAP; glyceraldehyde-3-phosphate; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; MWC; Monod, Wyman and Changeux; OAS; O; -acetylserine; OASS; O; -acetylserine sulfhydrylase; PEG; polyethylene glycol; PLP; pyridoxal 5′-phosphateProtein function; X-ray crystallography; Microspectrophotometry; Chromophoric protein
Protein crystal microspectrophotometry by Luca Ronda; Stefano Bruno; Stefano Bettati; Andrea Mozzarelli (pp. 734-741).
Single crystal microspectrophotometry has emerged as a valuable technique for monitoring molecular events that take place within protein crystals, thus tightly coupling structure to function. Absorption and fluorescence spectra, ligand binding affinities and kinetic constants can be determined, allowing i) the definition of the experimental conditions for X-ray crystallography experiments and their interpretation, ii) the assessment of whether crystal lattice forces have altered conformational equilibria, iii) the comparison with data obtained in solution. Microspectrophotometric measurements using oriented crystals and linearly polarized light are carried out usually off-line with respect to X-ray data collection and are aimed at an in- depth characterization of protein function in the crystal, leading to robust structure–function relationships. The power of this approach is highlighted by reporting a few case studies, including hemoglobins, pyridoxal 5′-phosphate-dependent enzymes and acetylcholinesterases. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.
Keywords: Abbreviations; GAP; glyceraldehyde-3-phosphate; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; MWC; Monod, Wyman and Changeux; OAS; O; -acetylserine; OASS; O; -acetylserine sulfhydrylase; PEG; polyethylene glycol; PLP; pyridoxal 5′-phosphateProtein function; X-ray crystallography; Microspectrophotometry; Chromophoric protein
Kinetic crystallography by Raman microscopy by Paul R. Carey; Yuanyuan Chen; Bo Gong; Matthew Kalp (pp. 742-749).
Raman spectra, obtained using a Raman microscope, offer a unique and incisive approach to follow interactions and reactions inside a single crystal under soak-in or soak-out conditions. The utility of this approach derives from the finding that the Raman spectra from single macromolecular crystals, under normal (non-resonance) conditions, are extremely stable, with a low “light background,” and provide ideal platforms for Raman difference spectroscopy. In turn, this allows the interrogation of sub-molecular changes in very large and complex macromolecular environments. There is often great synergy with X-ray crystallography, with the Raman spectroscopist providing crystallography colleagues with the best soak-in conditions to generate a targeted intermediate for flash freezing and X-ray analysis. On the other hand, X-ray structures at points along a reaction pathway provide invaluable benchmarks for interpreting the Raman data from populations seen by Raman to be changing in real-time. These principles will be illustrated by two reactions: the first involves a complex, branching reaction pathway underlying the inhibition of β-lactamases by clinically important pharmaceutical compounds, where different combinations of drug and enzyme function in different regions of the pathway. The second shows how temporal data can be derived for several events in the initiation step of RNA synthesis—more specifically, when one GTP molecule is joined to one ATP molecule to form a G∙A dimer in the active site of a 115,000Dalton crystalline RNA polymerase. Finally, we will summarize the extension of Raman microscopy to nucleic acid crystals and the information that has been obtained for RNA-based enzymes. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.
Keywords: Abbreviations; BC; binary complex; CCD; charge-coupled device; E166A; a deacylation-deficient variant of SHV-1 β-lactamase; HDV; hepatitis D virus; M69V; an inhibitor resistant variant of SHV-1 β-lactamase; NTP; nucleoside triphosphate; N*TP; nucleoside triphosphate in which the nucleic acid is completely labeled with; 13; C and; 15; N; PC; product complex; RNAP; RNA polymerase; SCI; substrate complex I; SCII; substrate complex II; SHV; class A β-lactamase of; K. pneumoniae; initially thought to be a “sulfhydryl variant” of the TEM enzyme; TC; transcarboxylase; WT; wild-typeRaman spectroscopy; Raman microscopy; Raman crystallography; Kinetic crystallography; β-Lactamase; RNA polymerase
Kinetic crystallography by Raman microscopy by Paul R. Carey; Yuanyuan Chen; Bo Gong; Matthew Kalp (pp. 742-749).
Raman spectra, obtained using a Raman microscope, offer a unique and incisive approach to follow interactions and reactions inside a single crystal under soak-in or soak-out conditions. The utility of this approach derives from the finding that the Raman spectra from single macromolecular crystals, under normal (non-resonance) conditions, are extremely stable, with a low “light background,” and provide ideal platforms for Raman difference spectroscopy. In turn, this allows the interrogation of sub-molecular changes in very large and complex macromolecular environments. There is often great synergy with X-ray crystallography, with the Raman spectroscopist providing crystallography colleagues with the best soak-in conditions to generate a targeted intermediate for flash freezing and X-ray analysis. On the other hand, X-ray structures at points along a reaction pathway provide invaluable benchmarks for interpreting the Raman data from populations seen by Raman to be changing in real-time. These principles will be illustrated by two reactions: the first involves a complex, branching reaction pathway underlying the inhibition of β-lactamases by clinically important pharmaceutical compounds, where different combinations of drug and enzyme function in different regions of the pathway. The second shows how temporal data can be derived for several events in the initiation step of RNA synthesis—more specifically, when one GTP molecule is joined to one ATP molecule to form a G∙A dimer in the active site of a 115,000Dalton crystalline RNA polymerase. Finally, we will summarize the extension of Raman microscopy to nucleic acid crystals and the information that has been obtained for RNA-based enzymes. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.
Keywords: Abbreviations; BC; binary complex; CCD; charge-coupled device; E166A; a deacylation-deficient variant of SHV-1 β-lactamase; HDV; hepatitis D virus; M69V; an inhibitor resistant variant of SHV-1 β-lactamase; NTP; nucleoside triphosphate; N*TP; nucleoside triphosphate in which the nucleic acid is completely labeled with; 13; C and; 15; N; PC; product complex; RNAP; RNA polymerase; SCI; substrate complex I; SCII; substrate complex II; SHV; class A β-lactamase of; K. pneumoniae; initially thought to be a “sulfhydryl variant” of the TEM enzyme; TC; transcarboxylase; WT; wild-typeRaman spectroscopy; Raman microscopy; Raman crystallography; Kinetic crystallography; β-Lactamase; RNA polymerase
Raman-assisted crystallography of biomolecules at the synchrotron: Instrumentation, methods and applications by John E. McGeehan; Dominique Bourgeois; Antoine Royant; Philippe Carpentier (pp. 750-759).
Raman spectroscopy is a powerful technique that, in recent years, has been successfully coupled to X-ray crystallography for the analysis of biological macromolecular systems. The complementarity between both techniques is illustrated at multiple stages, including sample preparation, data collection and structural interpretation with a mechanistic perspective. The current state of instrumentation is described, focusing on synchrotron based setups. Present and future applications of Raman microspectrophotometry are reviewed with reference to recent examples dealing with metallo-, photosensitive-, and redox-proteins. The added value of Raman microspectrophotometry to assess X-radiation damage is discussed, and its applicability to investigate crystalline DNA molecules is also emphasized. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.► Raman spectroscopy is an emerging technique at synchrotrons. ► Raman microspectrophotometry provides information highly complementary to macromolecular crystallography, shedding light on the mechanism of enzymes. ► Raman microspectrophotometry is a valuable non-invasive tool for quantifying radiation damage in macromolecular crystallography and providing the zero-dose point.
Keywords: Abbreviations; BM; bending magnet beamline; CCD; charge-coupled device; DFT; density functional theory; ESPT; excited state proton transfer; FMN; flavin mononucleotide; GFP; green fluorescent protein; ID; insertion device beamline; MASSIF; massively automated sample selection integrated facility; MX; macromolecular crystallography; NIR; near infra-red; PDB; protein databank; QM/MM; quantum mechanics/molecular mechanics; RaX; Raman-assisted crystallography; SOR; superoxide reductase; UV; ultra-violet; Vis; visible; XANES; X-ray Absorption Near Edge Structure SpectroscopyRaman spectroscopy; Macromolecular X-ray crystallography; Complementary methods; Raman-assisted crystallography (RaX); Synchrotron
Raman-assisted crystallography of biomolecules at the synchrotron: Instrumentation, methods and applications by John E. McGeehan; Dominique Bourgeois; Antoine Royant; Philippe Carpentier (pp. 750-759).
Raman spectroscopy is a powerful technique that, in recent years, has been successfully coupled to X-ray crystallography for the analysis of biological macromolecular systems. The complementarity between both techniques is illustrated at multiple stages, including sample preparation, data collection and structural interpretation with a mechanistic perspective. The current state of instrumentation is described, focusing on synchrotron based setups. Present and future applications of Raman microspectrophotometry are reviewed with reference to recent examples dealing with metallo-, photosensitive-, and redox-proteins. The added value of Raman microspectrophotometry to assess X-radiation damage is discussed, and its applicability to investigate crystalline DNA molecules is also emphasized. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.► Raman spectroscopy is an emerging technique at synchrotrons. ► Raman microspectrophotometry provides information highly complementary to macromolecular crystallography, shedding light on the mechanism of enzymes. ► Raman microspectrophotometry is a valuable non-invasive tool for quantifying radiation damage in macromolecular crystallography and providing the zero-dose point.
Keywords: Abbreviations; BM; bending magnet beamline; CCD; charge-coupled device; DFT; density functional theory; ESPT; excited state proton transfer; FMN; flavin mononucleotide; GFP; green fluorescent protein; ID; insertion device beamline; MASSIF; massively automated sample selection integrated facility; MX; macromolecular crystallography; NIR; near infra-red; PDB; protein databank; QM/MM; quantum mechanics/molecular mechanics; RaX; Raman-assisted crystallography; SOR; superoxide reductase; UV; ultra-violet; Vis; visible; XANES; X-ray Absorption Near Edge Structure SpectroscopyRaman spectroscopy; Macromolecular X-ray crystallography; Complementary methods; Raman-assisted crystallography (RaX); Synchrotron
Infrared protein crystallography by J. Timothy Sage; Yunbin Zhang; John McGeehan; Raimond B.G. Ravelli; Martin Weik; Jasper J. van Thor (pp. 760-777).
We consider the application of infrared spectroscopy to protein crystals, with particular emphasis on exploiting molecular orientation through polarization measurements on oriented single crystals. Infrared microscopes enable transmission measurements on individual crystals using either thermal or nonthermal sources, and can accommodate flow cells, used to measure spectral changes induced by exposure to soluble ligands, and cryostreams, used for measurements of flash-cooled crystals. Comparison of unpolarized infrared measurements on crystals and solutions probes the effects of crystallization and can enhance the value of the structural models refined from X-ray diffraction data by establishing solution conditions under which they are most relevant. Results on several proteins are consistent with similar equilibrium conformational distributions in crystal and solutions. However, the rates of conformational change are often perturbed. Infrared measurements also detect products generated by X-ray exposure, including CO2. Crystals with favorable symmetry exhibit infrared dichroism that enhances the synergy with X-ray crystallography. Polarized infrared measurements on crystals can distinguish spectral contributions from chemically similar sites, identify hydrogen bonding partners, and, in opportune situations, determine three-dimensional orientations of molecular groups. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.► Infrared spectroscopy: a versatile tool for investigating protein crystals. ► Instrumentation and applications are reviewed. ► Spectral comparison with solutions complements X-ray structural determination. ► Infrared dichroism distinguishes chemically similar sites. ► Products of radiation exposure contribute to the infrared signal.
Keywords: Infrared crystallography; Infrared dichroism; Radiation damage; Vibrational spectroscopy
Infrared protein crystallography by J. Timothy Sage; Yunbin Zhang; John McGeehan; Raimond B.G. Ravelli; Martin Weik; Jasper J. van Thor (pp. 760-777).
We consider the application of infrared spectroscopy to protein crystals, with particular emphasis on exploiting molecular orientation through polarization measurements on oriented single crystals. Infrared microscopes enable transmission measurements on individual crystals using either thermal or nonthermal sources, and can accommodate flow cells, used to measure spectral changes induced by exposure to soluble ligands, and cryostreams, used for measurements of flash-cooled crystals. Comparison of unpolarized infrared measurements on crystals and solutions probes the effects of crystallization and can enhance the value of the structural models refined from X-ray diffraction data by establishing solution conditions under which they are most relevant. Results on several proteins are consistent with similar equilibrium conformational distributions in crystal and solutions. However, the rates of conformational change are often perturbed. Infrared measurements also detect products generated by X-ray exposure, including CO2. Crystals with favorable symmetry exhibit infrared dichroism that enhances the synergy with X-ray crystallography. Polarized infrared measurements on crystals can distinguish spectral contributions from chemically similar sites, identify hydrogen bonding partners, and, in opportune situations, determine three-dimensional orientations of molecular groups. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.► Infrared spectroscopy: a versatile tool for investigating protein crystals. ► Instrumentation and applications are reviewed. ► Spectral comparison with solutions complements X-ray structural determination. ► Infrared dichroism distinguishes chemically similar sites. ► Products of radiation exposure contribute to the infrared signal.
Keywords: Infrared crystallography; Infrared dichroism; Radiation damage; Vibrational spectroscopy
Monitoring and validating active site redox states in protein crystals by Svetlana V. Antonyuk; Michael A. Hough (pp. 778-784).
High resolution protein crystallography using synchrotron radiation is one of the most powerful tools in modern biology. Improvements in resolution have arisen from the use of X-ray beamlines with higher brightness and flux and the development of advanced detectors. However, it is increasingly recognised that the benefits brought by these advances have an associated cost, namely deleterious effects of X-ray radiation on the sample (radiation damage). In particular, X-ray induced reduction and damage to redox centres has been shown to occur much more rapidly than other radiation damage effects, such as loss of resolution or damage to disulphide bridges. Selection of an appropriate combination of in-situ single crystal spectroscopies during crystallographic experiments, such as UV–visible absorption and X-ray absorption spectroscopy (XAFS), allows for effective monitoring of redox states in protein crystals in parallel with structure determination. Such approaches are also essential in cases where catalytic intermediate species are generated by exposure to the X-ray beam. In this article, we provide a number of examples in which multiple single crystal spectroscopies have been key to understanding the redox status of Fe and Cu centres in crystal structures. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.►Validation of redox states in crystal structures is an essential tool. ►Haem and Cu centres in protein crystals were monitored spectroscopically, in situ. ►These centres were shown to reduce rapidly in the X-ray beam. ►Spectroscopically validated structures of bacterioferritin and CuNiR were determined.
Keywords: Abbreviations; XAS; X-ray absorption spectroscopy; PX; Protein crystallography; PIXE; Proton induced X-ray emission; XRF; X-ray fluorescence; PDB; RCSB Protein Data Bank; Ec; Bf; apo bacterioferritin from; E. coli; Ac; NiR; Cu nitrite reductase from; Achromobacter cycloclastes; SLS; Swiss Light SourceRedox protein; Metalloprotein; Structure validation; Combined method; XAS; Spectroscopy
Monitoring and validating active site redox states in protein crystals by Svetlana V. Antonyuk; Michael A. Hough (pp. 778-784).
High resolution protein crystallography using synchrotron radiation is one of the most powerful tools in modern biology. Improvements in resolution have arisen from the use of X-ray beamlines with higher brightness and flux and the development of advanced detectors. However, it is increasingly recognised that the benefits brought by these advances have an associated cost, namely deleterious effects of X-ray radiation on the sample (radiation damage). In particular, X-ray induced reduction and damage to redox centres has been shown to occur much more rapidly than other radiation damage effects, such as loss of resolution or damage to disulphide bridges. Selection of an appropriate combination of in-situ single crystal spectroscopies during crystallographic experiments, such as UV–visible absorption and X-ray absorption spectroscopy (XAFS), allows for effective monitoring of redox states in protein crystals in parallel with structure determination. Such approaches are also essential in cases where catalytic intermediate species are generated by exposure to the X-ray beam. In this article, we provide a number of examples in which multiple single crystal spectroscopies have been key to understanding the redox status of Fe and Cu centres in crystal structures. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.►Validation of redox states in crystal structures is an essential tool. ►Haem and Cu centres in protein crystals were monitored spectroscopically, in situ. ►These centres were shown to reduce rapidly in the X-ray beam. ►Spectroscopically validated structures of bacterioferritin and CuNiR were determined.
Keywords: Abbreviations; XAS; X-ray absorption spectroscopy; PX; Protein crystallography; PIXE; Proton induced X-ray emission; XRF; X-ray fluorescence; PDB; RCSB Protein Data Bank; Ec; Bf; apo bacterioferritin from; E. coli; Ac; NiR; Cu nitrite reductase from; Achromobacter cycloclastes; SLS; Swiss Light SourceRedox protein; Metalloprotein; Structure validation; Combined method; XAS; Spectroscopy
How different oxidation states of crystalline myoglobin are influenced by X-rays by Hans-Petter Hersleth; K. Kristoffer Andersson (pp. 785-796).
X-ray induced radiation damage of protein crystals is well known to occur even at cryogenic temperatures. Redox active sites like metal sites seem especially vulnerable for these radiation-induced reductions. It is essential to know correctly the oxidation state of metal sites in protein crystal structures to be able to interpret the structure–function relation. Through previous structural studies, we have tried to characterise and understand the reactions between myoglobin and peroxides. These reaction intermediates are relevant because myoglobin is proposed to take part as scavenger of reactive oxygen species during oxidative stress, and because these intermediates are similar among the haem peroxidases and oxygenases. We have in our previous studies shown that these different myoglobin states are influenced by the X-rays used. In this study, we have in detail investigated the impact that X-rays have on these different oxidation states of myoglobin. An underlying goal has been to find a way to be able to determine mostly unreduced states. We have by using single-crystal light absorption spectroscopy found that the different oxidation states of myoglobin are to a different extent influenced by the X-rays (e.g. ferric FeIII myoglobin is faster reduced than ferryl FeIV═O myoglobin). We observe that the higher oxidation states are not reduced to normal ferrous FeII or ferric FeIII states, but end up in some intermediate and possibly artificial states. For ferric myoglobin, it seems that annealing of the radiation-induced/reduced state can reversibly more or less give the starting point (ferric myoglobin). Both scavengers and different dose-rates might influence to which extent the different states are affected by the X-rays. Our study shows that it is essential to do a time/dose monitoring of the influence X-rays have on each specific redox-state with spectroscopic techniques like single-crystal light absorption spectroscopy. This will determine to which extent you can collect X-ray diffraction data on your crystal before it becomes too heavily influenced/reduced by X-rays. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.
Keywords: Abbreviations; Mb; myoglobin; ESRF; European Synchrotron Radiation Facility; SNBL; Swiss–Norwegian Beam Line; SLS; Swiss Light Source; metMb; myoglobin in its oxidised ferric form; met; metmyoglobin; cmpII; myoglobin compound II; cmpIII; myoglobin compound III; rRaman; resonance RamanRadiation damage; Metalloprotein; X-ray; Crystal structure; Spectroscopy; Peroxidase
How different oxidation states of crystalline myoglobin are influenced by X-rays by Hans-Petter Hersleth; K. Kristoffer Andersson (pp. 785-796).
X-ray induced radiation damage of protein crystals is well known to occur even at cryogenic temperatures. Redox active sites like metal sites seem especially vulnerable for these radiation-induced reductions. It is essential to know correctly the oxidation state of metal sites in protein crystal structures to be able to interpret the structure–function relation. Through previous structural studies, we have tried to characterise and understand the reactions between myoglobin and peroxides. These reaction intermediates are relevant because myoglobin is proposed to take part as scavenger of reactive oxygen species during oxidative stress, and because these intermediates are similar among the haem peroxidases and oxygenases. We have in our previous studies shown that these different myoglobin states are influenced by the X-rays used. In this study, we have in detail investigated the impact that X-rays have on these different oxidation states of myoglobin. An underlying goal has been to find a way to be able to determine mostly unreduced states. We have by using single-crystal light absorption spectroscopy found that the different oxidation states of myoglobin are to a different extent influenced by the X-rays (e.g. ferric FeIII myoglobin is faster reduced than ferryl FeIV═O myoglobin). We observe that the higher oxidation states are not reduced to normal ferrous FeII or ferric FeIII states, but end up in some intermediate and possibly artificial states. For ferric myoglobin, it seems that annealing of the radiation-induced/reduced state can reversibly more or less give the starting point (ferric myoglobin). Both scavengers and different dose-rates might influence to which extent the different states are affected by the X-rays. Our study shows that it is essential to do a time/dose monitoring of the influence X-rays have on each specific redox-state with spectroscopic techniques like single-crystal light absorption spectroscopy. This will determine to which extent you can collect X-ray diffraction data on your crystal before it becomes too heavily influenced/reduced by X-rays. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.
Keywords: Abbreviations; Mb; myoglobin; ESRF; European Synchrotron Radiation Facility; SNBL; Swiss–Norwegian Beam Line; SLS; Swiss Light Source; metMb; myoglobin in its oxidised ferric form; met; metmyoglobin; cmpII; myoglobin compound II; cmpIII; myoglobin compound III; rRaman; resonance RamanRadiation damage; Metalloprotein; X-ray; Crystal structure; Spectroscopy; Peroxidase
Hemoglobin–ligand binding: Understanding Hb function and allostery on atomic level by Martin K. Safo; Mostafa H. Ahmed; Mohini S. Ghatge; Telih Boyiri (pp. 797-809).
The major physiological function of hemoglobin (Hb) is to bind oxygen in the lungs and deliver it to the tissues. This function is regulated and/or made efficient by endogenous heterotropic effectors. A number of synthetic molecules also bind to Hb to alter its allosteric activity. Our purpose is to review the current state of Hb structure and function that involves ensemble of tense and relaxed hemoglobin states and the dynamic equilibrium of the multistate due to the binding of endogenous heterotropic or synthetic allosteric effectors. The review also discusses the atomic interactions of synthetic ligands with the function or altered allosteric function of Hb that could be potentially harnessed for the treatment of diseases. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.► Hemoglobin structure and function involves ensemble of tense and relaxed states. ► Effectors can bind to the same site in Hb but produce opposite allosteric effects. ► Aromatic aldehydes can increase the fraction of the more soluble oxygenated sickle Hb. ► Allosteric effectors of Hb can increase oxygen delivery to tissues. ► αArg141, βTrp37 and G α-helix residues are important in Hb quaternary constraint.
Keywords: Deoxygenated; Oxygenated; Hemoglobin; Relaxed, tense; Allosteric
Hemoglobin–ligand binding: Understanding Hb function and allostery on atomic level by Martin K. Safo; Mostafa H. Ahmed; Mohini S. Ghatge; Telih Boyiri (pp. 797-809).
The major physiological function of hemoglobin (Hb) is to bind oxygen in the lungs and deliver it to the tissues. This function is regulated and/or made efficient by endogenous heterotropic effectors. A number of synthetic molecules also bind to Hb to alter its allosteric activity. Our purpose is to review the current state of Hb structure and function that involves ensemble of tense and relaxed hemoglobin states and the dynamic equilibrium of the multistate due to the binding of endogenous heterotropic or synthetic allosteric effectors. The review also discusses the atomic interactions of synthetic ligands with the function or altered allosteric function of Hb that could be potentially harnessed for the treatment of diseases. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.► Hemoglobin structure and function involves ensemble of tense and relaxed states. ► Effectors can bind to the same site in Hb but produce opposite allosteric effects. ► Aromatic aldehydes can increase the fraction of the more soluble oxygenated sickle Hb. ► Allosteric effectors of Hb can increase oxygen delivery to tissues. ► αArg141, βTrp37 and G α-helix residues are important in Hb quaternary constraint.
Keywords: Deoxygenated; Oxygenated; Hemoglobin; Relaxed, tense; Allosteric
Structural characterization of a group II 2/2 hemoglobin from the plant pathogen Agrobacterium tumefaciens by Alessandra Pesce; Marco Nardini; Marie LaBarre; Christian Richard; Jonathan B. Wittenberg; Beatrice A. Wittenberg; Michel Guertin; Martino Bolognesi (pp. 810-816).
Within the 2/2 hemoglobin sub-family, no group II 2/2Hbs from proteobacteria have been so far studied. Here we present the first structural characterization of a group II 2/2Hb from the soil and phytopathogenic bacterium Agrobacterium tumefaciens ( At-2/2HbO). The crystal structure of ferric At-2/2HbO (reported at 2.1Å resolution) shows the location of specific/unique heme distal site residues (e.g., His(42)CD1, a residue distinctive of proteobacteria group II 2/2Hbs) that surround a heme-liganded water molecule. A highly intertwined hydrogen-bonded network, involving residues Tyr(26)B10, His(42)CD1, Ser(49)E7, Trp(93)G8, and three distal site water molecules, stabilizes the heme-bound ligand. Such a structural organization suggests a path for diatomic ligand diffusion to/from the heme. Neither a similar distal site structuring effect nor the presence of distal site water molecules has been so far observed in group I and group III 2/2Hbs, thus adding new distinctive information to the complex picture of currently available 2/2Hb structural and functional data. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.► Agrobacterium tumefaciens 2/2 hemoglobin three-dimensional structure, aquo-met form. ► A hydrogen-bonded network of water molecules in the heme distal site cavity. ► Heme distal site residue variability among group I, II and III 2/2 hemoglobins. ► Diatomic ligand diffusion path to the heme in A. tumefaciens 2/2HbO. ► HisCD1 vs. the conserved PheCD1 residue in A. tumefaciens 2/2HbO.
Keywords: Abbreviations; 2/2Hb; 2-on-2 hemoglobin; Mt; -2/2HbO; Mycobacterium tuberculosis; 2/2 hemoglobin O; Tf; -2/2HbO; Thermobifida fusca; 2/2 hemoglobin O; Bs; -2/2HbO; Bacillus subtilis; 2/2 hemoglobin O; Gs; -2/2HbO; Geobacillus stearothermophilus; 2/2 hemoglobin O; At; -2/2HbO; Agrobacterium tumefaciens; 2/2 hemoglobin O; Ce; -2/2HbN; Chlamydomonas eugametos; hemoglobin N; Cj; -2/2HbP; Campylobacter jejuni; 2/2 hemoglobin P2/2 Hemoglobin; Truncated hemoglobin; Globin fold; Heme stabilization; Diatomic ligand recognition
Structural characterization of a group II 2/2 hemoglobin from the plant pathogen Agrobacterium tumefaciens by Alessandra Pesce; Marco Nardini; Marie LaBarre; Christian Richard; Jonathan B. Wittenberg; Beatrice A. Wittenberg; Michel Guertin; Martino Bolognesi (pp. 810-816).
Within the 2/2 hemoglobin sub-family, no group II 2/2Hbs from proteobacteria have been so far studied. Here we present the first structural characterization of a group II 2/2Hb from the soil and phytopathogenic bacterium Agrobacterium tumefaciens ( At-2/2HbO). The crystal structure of ferric At-2/2HbO (reported at 2.1Å resolution) shows the location of specific/unique heme distal site residues (e.g., His(42)CD1, a residue distinctive of proteobacteria group II 2/2Hbs) that surround a heme-liganded water molecule. A highly intertwined hydrogen-bonded network, involving residues Tyr(26)B10, His(42)CD1, Ser(49)E7, Trp(93)G8, and three distal site water molecules, stabilizes the heme-bound ligand. Such a structural organization suggests a path for diatomic ligand diffusion to/from the heme. Neither a similar distal site structuring effect nor the presence of distal site water molecules has been so far observed in group I and group III 2/2Hbs, thus adding new distinctive information to the complex picture of currently available 2/2Hb structural and functional data. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.► Agrobacterium tumefaciens 2/2 hemoglobin three-dimensional structure, aquo-met form. ► A hydrogen-bonded network of water molecules in the heme distal site cavity. ► Heme distal site residue variability among group I, II and III 2/2 hemoglobins. ► Diatomic ligand diffusion path to the heme in A. tumefaciens 2/2HbO. ► HisCD1 vs. the conserved PheCD1 residue in A. tumefaciens 2/2HbO.
Keywords: Abbreviations; 2/2Hb; 2-on-2 hemoglobin; Mt; -2/2HbO; Mycobacterium tuberculosis; 2/2 hemoglobin O; Tf; -2/2HbO; Thermobifida fusca; 2/2 hemoglobin O; Bs; -2/2HbO; Bacillus subtilis; 2/2 hemoglobin O; Gs; -2/2HbO; Geobacillus stearothermophilus; 2/2 hemoglobin O; At; -2/2HbO; Agrobacterium tumefaciens; 2/2 hemoglobin O; Ce; -2/2HbN; Chlamydomonas eugametos; hemoglobin N; Cj; -2/2HbP; Campylobacter jejuni; 2/2 hemoglobin P2/2 Hemoglobin; Truncated hemoglobin; Globin fold; Heme stabilization; Diatomic ligand recognition
X-ray crystallography, mass spectrometry and single crystal microspectrophotometry: A multidisciplinary characterization of catechol 1,2 dioxygenase by Chiara Micalella; Sara Martignon; Stefano Bruno; Barbara Pioselli; Raffaella Caglio; Francesca Valetti; Enrica Pessione; Carlo Giunta; Menico Rizzi (pp. 817-823).
Intradiol-cleaving catechol 1,2 dioxygenases are Fe(III) dependent enzymes that act on catechol and substituted catechols, including chlorocatechols pollutants, by inserting molecular oxygen in the aromatic ring. Members of this class are the object of intense biochemical investigations aimed at the understanding of their catalytic mechanism, particularly for designing mutants with selected catalytic properties. We report here an in depth investigation of catechol 1,2 dioxygenase IsoB from Acinetobacter radioresistens LMG S13 and its A72G and L69A mutants. By applying a multidisciplinary approach that includes high resolution X-rays crystallography, mass spectrometry and single crystal microspectrophotometry, we characterised the phospholipid bound to the enzyme and provided a structural framework to understand the inversion of substrate specificity showed by the mutants. Our results might be of help for the rational design of enzyme mutants showing a biotechnologically relevant substrate specificity, particularly to be used in bioremediation. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.► Microspectrophotometric analysis reveals that Acinetobacter radioresistens catechol 1,2-dioxygenase behaves in a similar way in solution and in the crystalline state with respect to both Fe(III) coordination and ligand binding properties. ► By combining X-ray crystallography and mass spectrometry, the phospholipid tightly bound at the dimer interface was identified to be either a glycerophosphoinositol or a glycerophosphoinositol monophosphate with hydrophobic tail C18:0/C16:1(9Z). ► Crystallographic investigation provides an explanation for the inversion of specificity shown by the L69A and A72G variants in catechol 1,2-dioxygenase from Acinetobacter radioresistens.
Keywords: Abbreviations; 1,2-CTD; Catechol 1,2 dioxygenase; Ar-1,2-CTD; Catechol 1,2 dioxygenase from A. Radioresistens S13; 1,2-CCD; Chlorocatechol 1,2 dioxygenase; PIP; Phosphoinositol monophosphate; IPTG; Isopropyl β-D-thiogalactopyranosideCatechol dioxygenase; Non-Fe oxygenase; Microspectrophotometry; X-ray crystallography; Bioremediation
X-ray crystallography, mass spectrometry and single crystal microspectrophotometry: A multidisciplinary characterization of catechol 1,2 dioxygenase by Chiara Micalella; Sara Martignon; Stefano Bruno; Barbara Pioselli; Raffaella Caglio; Francesca Valetti; Enrica Pessione; Carlo Giunta; Menico Rizzi (pp. 817-823).
Intradiol-cleaving catechol 1,2 dioxygenases are Fe(III) dependent enzymes that act on catechol and substituted catechols, including chlorocatechols pollutants, by inserting molecular oxygen in the aromatic ring. Members of this class are the object of intense biochemical investigations aimed at the understanding of their catalytic mechanism, particularly for designing mutants with selected catalytic properties. We report here an in depth investigation of catechol 1,2 dioxygenase IsoB from Acinetobacter radioresistens LMG S13 and its A72G and L69A mutants. By applying a multidisciplinary approach that includes high resolution X-rays crystallography, mass spectrometry and single crystal microspectrophotometry, we characterised the phospholipid bound to the enzyme and provided a structural framework to understand the inversion of substrate specificity showed by the mutants. Our results might be of help for the rational design of enzyme mutants showing a biotechnologically relevant substrate specificity, particularly to be used in bioremediation. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.► Microspectrophotometric analysis reveals that Acinetobacter radioresistens catechol 1,2-dioxygenase behaves in a similar way in solution and in the crystalline state with respect to both Fe(III) coordination and ligand binding properties. ► By combining X-ray crystallography and mass spectrometry, the phospholipid tightly bound at the dimer interface was identified to be either a glycerophosphoinositol or a glycerophosphoinositol monophosphate with hydrophobic tail C18:0/C16:1(9Z). ► Crystallographic investigation provides an explanation for the inversion of specificity shown by the L69A and A72G variants in catechol 1,2-dioxygenase from Acinetobacter radioresistens.
Keywords: Abbreviations; 1,2-CTD; Catechol 1,2 dioxygenase; Ar-1,2-CTD; Catechol 1,2 dioxygenase from A. Radioresistens S13; 1,2-CCD; Chlorocatechol 1,2 dioxygenase; PIP; Phosphoinositol monophosphate; IPTG; Isopropyl β-D-thiogalactopyranosideCatechol dioxygenase; Non-Fe oxygenase; Microspectrophotometry; X-ray crystallography; Bioremediation
Structure and single crystal spectroscopy of Green Fluorescent Proteins by Stefano Bettati; Elisa Pasqualetto; Graziano Lolli; Barbara Campanini; Roberto Battistutta (pp. 824-833).
Usually, spectroscopic data on proteins in solution are interpreted at molecular level on the basis of the three-dimensional structures determined in the crystalline state. While it is widely recognized that the protein crystal structures are reliable models for the solution 3D structures, nevertheless it is also clear that sometimes the crystallization process can introduce some “artifacts” that can make difficult or even flaw the attempt to correlate the properties in solution with those in the crystalline state. In general, therefore, it would be desirable to identify some sort of control. In the case of the spectroscopic properties of proteins, the most straightforward check is to acquire data not only in solution but also on the crystals. In this regard, the Green Fluorescent Protein (GFP) is an interesting case in that a massive quantity of data correlating the spectroscopic properties in solution with the structural information in the crystalline state is available in literature. Despite that, a relatively limited amount of spectroscopic studies on single crystals of GFP or related FPs have been described. Here we review and discuss the main spectroscopic (in solution) and structural (in crystals) studies performed on the GFP and related fluorescent proteins, together with the spectroscopic analyses on various FPs members in the crystalline state. One main conclusion is that “in cristallo” spectroscopic studies are useful in providing new opportunities for gathering information not available in solution and are highly recommended to reliably correlate solution properties with structural features. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.
Keywords: Abbreviations; wtGFP; wild-type; Aequorea victoria; Green Fluorescent Protein; CPs; chromoproteins; FPs; fluorescent proteins; ESPT; excited state proton transfer; FRET; Förster resonance energy transfer; p-HBI; 4-(p-hydroxybenzylidene)-5-imidazolinone, wtGFP chromophore; ChrOH and ChrO; −; GFP chromophore in the protonated and deprotonated states, respectively; PEG; polyethylene glycolGreen fluorescent protein; GFP; ESPT; Spectroscopy; Protein crystals
Structure and single crystal spectroscopy of Green Fluorescent Proteins by Stefano Bettati; Elisa Pasqualetto; Graziano Lolli; Barbara Campanini; Roberto Battistutta (pp. 824-833).
Usually, spectroscopic data on proteins in solution are interpreted at molecular level on the basis of the three-dimensional structures determined in the crystalline state. While it is widely recognized that the protein crystal structures are reliable models for the solution 3D structures, nevertheless it is also clear that sometimes the crystallization process can introduce some “artifacts” that can make difficult or even flaw the attempt to correlate the properties in solution with those in the crystalline state. In general, therefore, it would be desirable to identify some sort of control. In the case of the spectroscopic properties of proteins, the most straightforward check is to acquire data not only in solution but also on the crystals. In this regard, the Green Fluorescent Protein (GFP) is an interesting case in that a massive quantity of data correlating the spectroscopic properties in solution with the structural information in the crystalline state is available in literature. Despite that, a relatively limited amount of spectroscopic studies on single crystals of GFP or related FPs have been described. Here we review and discuss the main spectroscopic (in solution) and structural (in crystals) studies performed on the GFP and related fluorescent proteins, together with the spectroscopic analyses on various FPs members in the crystalline state. One main conclusion is that “in cristallo” spectroscopic studies are useful in providing new opportunities for gathering information not available in solution and are highly recommended to reliably correlate solution properties with structural features. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.
Keywords: Abbreviations; wtGFP; wild-type; Aequorea victoria; Green Fluorescent Protein; CPs; chromoproteins; FPs; fluorescent proteins; ESPT; excited state proton transfer; FRET; Förster resonance energy transfer; p-HBI; 4-(p-hydroxybenzylidene)-5-imidazolinone, wtGFP chromophore; ChrOH and ChrO; −; GFP chromophore in the protonated and deprotonated states, respectively; PEG; polyethylene glycolGreen fluorescent protein; GFP; ESPT; Spectroscopy; Protein crystals
Exploring methionine γ-lyase structure-function relationship via microspectrophotometry and X-ray crystallography by Luca Ronda; Natalia P. Bazhulina; Elena A. Morozova; Svetlana V. Revtovich; Vladimir O. Chekhov; Alexei D. Nikulin; Tatyana V. Demidkina; Andrea Mozzarelli (pp. 834-842).
Pyridoxal 5’-phosphate (PLP) dependent methionine γ-lyase catalyzes the breakdown of L-methionine to α-ketobutyric acid, methanethiol and ammonia. This enzyme, present in anaerobic microorganisms, has biomedical interest both for its activity as antitumor agent, depleting methionine supply in methionine-dependent cancers, and as target in the treatment of human pathogen infections, activating the pro-drug trifluoromethionine. To validate the structure of the enzyme from Citrobacter freundii, crystallized from monomethyl ether polyethylene glycol 2000, for the development of lead compounds, the reactivity of the crystalline enzyme towards L-methionine, substrate analogs and inhibitors was determined by polarized absorption microspectrophotometry. Spectral data were also collected for enzyme crystals, grown in monomethyl ether polyethylene glycol 2000 in the presence of ammonium sulfate. The three-dimensional structure of these enzyme crystals, solved at 1.65Å resolution with Rfree 23.2%, revealed the surprising absence of the aldimine bond between the active site Lys210 and PLP. Different hypothesis are proposed and discussed in the light of spectral and structural data, pointing out to the relevance of the complementarity between X-ray crystallography and single crystal spectroscopy for the understanding of biological mechanisms at molecular level. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.
Keywords: Abbreviations; PLP; pyridoxal 5’-phosphate; MMEPEG; polyethylene glycol monomethyl ether; PR; polarization ratioMethionine γ-lyase; Pyridoxal 5’-phosphate; Single crystal spectroscopy; Catalytic intermediates; Three-dimensional structure
Exploring methionine γ-lyase structure-function relationship via microspectrophotometry and X-ray crystallography by Luca Ronda; Natalia P. Bazhulina; Elena A. Morozova; Svetlana V. Revtovich; Vladimir O. Chekhov; Alexei D. Nikulin; Tatyana V. Demidkina; Andrea Mozzarelli (pp. 834-842).
Pyridoxal 5’-phosphate (PLP) dependent methionine γ-lyase catalyzes the breakdown of L-methionine to α-ketobutyric acid, methanethiol and ammonia. This enzyme, present in anaerobic microorganisms, has biomedical interest both for its activity as antitumor agent, depleting methionine supply in methionine-dependent cancers, and as target in the treatment of human pathogen infections, activating the pro-drug trifluoromethionine. To validate the structure of the enzyme from Citrobacter freundii, crystallized from monomethyl ether polyethylene glycol 2000, for the development of lead compounds, the reactivity of the crystalline enzyme towards L-methionine, substrate analogs and inhibitors was determined by polarized absorption microspectrophotometry. Spectral data were also collected for enzyme crystals, grown in monomethyl ether polyethylene glycol 2000 in the presence of ammonium sulfate. The three-dimensional structure of these enzyme crystals, solved at 1.65Å resolution with Rfree 23.2%, revealed the surprising absence of the aldimine bond between the active site Lys210 and PLP. Different hypothesis are proposed and discussed in the light of spectral and structural data, pointing out to the relevance of the complementarity between X-ray crystallography and single crystal spectroscopy for the understanding of biological mechanisms at molecular level. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.
Keywords: Abbreviations; PLP; pyridoxal 5’-phosphate; MMEPEG; polyethylene glycol monomethyl ether; PR; polarization ratioMethionine γ-lyase; Pyridoxal 5’-phosphate; Single crystal spectroscopy; Catalytic intermediates; Three-dimensional structure