ABSTRACTHigh-resolution biomacromolecular structure determination is essential to better understa... more ABSTRACTHigh-resolution biomacromolecular structure determination is essential to better understand protein function and dynamics. Serial crystallography is an emerging structural biology technique which has fundamental limitations due to either sample volume requirements or immediate access to the competitive X-ray beamtime. Obtaining a high volume of well-diffracting, sufficient-size crystals while mitigating radiation damage remains a critical bottleneck of serial crystallography. As an alternative, we introduce the plate-reader module adapted for using a 72-well Terasaki plate for biomacromolecule structure determination at a convenience of a home X-ray source. We also present the first ambient temperature lysozyme structure determined at the Turkish Light Source (Turkish DeLight). The complete dataset was collected in 18.5 mins with resolution extending to 2.39 Å and 100% completeness. Combined with our previous cryogenic structure (PDB ID: 7Y6A), the ambient temperature struct...
X-ray crystallography is a robust and powerful structural biology technique that provides high-re... more X-ray crystallography is a robust and powerful structural biology technique that provides high-resolution atomic structures of biomacromolecules. Scientists use this technique to unravel mechanistic and structural details of biological macromolecules (e.g. proteins, nucleic acids, protein complexes, protein-nucleic acid complexes, or large biological compartments). Since its inception, single-crystal cryo-crystallography has never been performed in Türkiye due to the lack of a single-crystal X-ray diffractometer. The X-ray diffraction facility recently established at the University of Health Sciences, Istanbul, Türkiye will enable Turkish and international researchers to easily perform high-resolution structural analysis of biomacromolecules from single crystals. Here, we describe the technical and practical outlook of a state-of-the-art home-source X-ray, using lysozyme as a model protein. The methods and practice described in this article can be applied to any biological sample fo...
The biological route for nitrogen gas entering the biosphere is reduction to ammonia by the nitro... more The biological route for nitrogen gas entering the biosphere is reduction to ammonia by the nitrogenase enzyme, which is inactivated by oxygen. Three types of nitrogenase exist, the least studied of which is the iron-only nitrogenase. The Anf3 protein in the bacterium Rhodobacter capsulatus is essential for diazotrophic (i.e. nitrogen-fixing) growth with the iron-only nitrogenase, but its enzymatic activity and function are unknown. Here, we biochemically and structurally characterize Anf3 from the model diazotrophic bacterium Azotobacter vinelandii. Determining the Anf3 crystal structure to atomic resolution, we observed that it is a dimeric flavocytochrome with an unusually close interaction between the heme and the flavin adenine dinucleotide cofactors. Measuring the reduction potentials by spectroelectrochemical redox titration, we observed values of-420 ± 10 mV and-330 ± 10 mV for the two FAD potentials and-340 ± 1 mV for the heme. We further show that Anf3 accepts electrons from spinach ferredoxin and that Anf3 consumes oxygen without generating superoxide or hydrogen peroxide. We predict that Anf3 protects the iron-only nitrogenase from oxygen inactivation by functioning as an oxidase in respiratory protection, with flavodoxin or ferredoxin as the physiological electron donors.
Microorganisms use carboxylase enzymes to form new carbon-carbon bonds by introducing carbon diox... more Microorganisms use carboxylase enzymes to form new carbon-carbon bonds by introducing carbon dioxide gas (CO2) or its hydrated form, bicarbonate (HCO3(-)), into target molecules. Acetone carboxylases (ACs) catalyze the conversion of substrates acetone and HCO3(-) to form the product acetoacetate. Many bicarbonate-incorporating carboxylases rely on the organic cofactor biotin for the activation of bicarbonate. ACs contain metal ions but not organic cofactors, and use ATP to activate substrates through phosphorylation. How the enzyme coordinates these phosphorylation events and new C-C bond formation in the absence of biotin has remained a mystery since these enzymes were discovered. The first structural rationale for acetone carboxylation is presented here, focusing on the 360 kDa (αβγ)2 heterohexameric AC from Xanthobacter autotrophicus in the ligand-free, AMP-bound, and acetate coordinated states. These structures suggest successive steps in a catalytic cycle revealing that AC unde...
The dual-function fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) in cyanobacteria car... more The dual-function fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) in cyanobacteria carries out two activities in the Calvin cycle. Structures of this enzyme from the cyanobacterium Synechocystis sp. PCC 6803 exist, but only with adenosine monophosphate (AMP) or fructose-1,6-bisphosphate and AMP bound. The mechanisms which control both selectivity between the two sugars and the structural mechanisms for redox control are still unresolved. Here, the structure of the dual-function FBP/SBPase from the thermophilic cyanobacterium Thermosynechococcus elongatus is presented with sedoheptulose-7-phosphate bound and in the absence of AMP. The structure is globally very similar to the Synechocystis sp. PCC 6803 enzyme, but highlights features of selectivity at the active site and loop ordering at the AMP-binding site. Understanding the selectivity and control of this enzyme is critical for understanding the Calvin cycle in cyanobacteria and for possible biotechnological application...
The goal of this study was to develop and validate a liquid chromatography (LC) method for the ra... more The goal of this study was to develop and validate a liquid chromatography (LC) method for the rapid determination of dipyrone (metamizole sodium) in solid and liquid formulations. Samples were extracted with water and filtered prior to LC analysis. The analytical procedure involved reversed phase LC with a Zorbax SB C 18 column (5 m particle size, 4.6 mm × 250 mm i.d.), isoctratic methanol/water (80/20, v/v) mobile phase with UV detection at 254 nm. The flow rate was 1 mL min -1 , injection volume was 100 L and retention time of dipyrone was 3.3 min. The method was validated with respect to linearity, precision and accuracy. Calibration curves were linear in the range 0.5-100 g mL -1 for analyte and the recoveries at three levels (10, 15 and 25 g mL -1 ) ranged from 93 to 100%. All results for dipyrone were confirmed by liquid chromatography-mass spectrometry (LC-MS) and 1 H nuclear magnetic resonance (NMR) spectroscopy. Due to its simplicity and accuracy, the assay method is suitable for routine analysis of both solid and liquid formulations and seven retail samples were analyzed using the validated method.
In plants, carbon dioxide is fixed via the Calvin cycle in a tightly regulated process. Key to th... more In plants, carbon dioxide is fixed via the Calvin cycle in a tightly regulated process. Key to this regulation is the conditionally disordered protein CP12. CP12 forms a complex with two Calvin cycle enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), inhibiting their activities. The mode of CP12 action was unknown. By solving crystal structures of CP12 bound to GAPDH, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide pre-orders CP12 prior to binding the PRK active site. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our model explains how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation.One Sentence SummaryHow plants turn off carbon fixation in the dark.
Transesterification by methanol and interesterification by methyl acetate were performed simultan... more Transesterification by methanol and interesterification by methyl acetate were performed simultaneously, and the advantages of using methyl acetate with methanol were investigated in the enzymatic production of methyl ester from tributyrin. It was observed that 100% excess amount (1:6 mol ratio) of methanol/methyl acetate makes no negative effect on the enzyme activity. The initial reaction rate decreases as the tributyrin concentration increases. Consequently, tributyrin causes an inhibition at high concentrations. When the tributyrin concentration was used above the stoichiometric ratio (1:3 mol ratio), it decreased the activity of the enzyme. Michaelis-Menten parameters (K m) for tributyrin and methanol/methyl acetate were calculated as 0.1 and 50 M, respectively. An uncompetitive substrate inhibition constant for tributyrin was determined as 22.42 M. Experimental results were found to correlate well with the results of the kinetic model according to the ping-pong bi-bi mechanism. High conversions up to 90% were observed in a semicontinuous fluidized bed with low enzyme levels (1%). Conversions decreased with increasing flow rates.
Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the proton-coupled four-electron reduc... more Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the proton-coupled four-electron reduction 27 of biliverdin IXa's two vinyl groups to produce phycocyanobilin, an essential chromophore for phyto-28 chromes, cyanobacteriochromes and phycobiliproteins. Previous site directed mutagenesis studies indi-29 cated that the fully conserved residue His74 plays a critical role in the H-bonding network that permits 30 proton transfer. Here, we exploit X-ray crystallography, enzymology and molecular dynamics simulations 31 to understand the functional role of this invariant histidine. The structures of the H74A, H74E and H74Q 32 variants of PcyA reveal that a ''conserved'' buried water molecule that bridges His74 and catalytically 33 essential His88 is not required for activity. Despite distinct conformations of Glu74 and Gln74 in theH74E 34 and H74Q variants, both retain reasonable activity while the H74A variant is inactive, suggesting smaller 35 residues may generate cavities that increase flexibility, thereby reducing enzymatic activity. Molecular 36 dynamic simulations further reveal that the crucial active site residue Asp105 is more dynamic in 37 H74A compared to wild-type PcyA and the two other His74 variants, supporting the conclusion that 38 the Ala74 mutation has increased the flexibility of the active site.
Proceedings of the National Academy of Sciences of the United States of America, Sep 13, 2016
The ability to design and construct structures with atomic level precision is one of the key goal... more The ability to design and construct structures with atomic level precision is one of the key goals of nanotechnology. Proteins offer an attractive target for atomic design because they can be synthesized chemically or biologically and can self-assemble. However, the generalized protein folding and design problem is unsolved. One approach to simplifying the problem is to use a repetitive protein as a scaffold. Repeat proteins are intrinsically modular, and their folding and structures are better understood than large globular domains. Here, we have developed a class of synthetic repeat proteins based on the pentapeptide repeat family of beta-solenoid proteins. We have constructed length variants of the basic scaffold and computationally designed de novo loops projecting from the scaffold core. The experimentally solved 3.56-Å resolution crystal structure of one designed loop matches closely the designed hairpin structure, showing the computational design of a backbone extension onto ...
Transesterification by methanol and interesterification by methyl acetate were performed simultan... more Transesterification by methanol and interesterification by methyl acetate were performed simultaneously, and the advantages of using methyl acetate with methanol were investigated in the enzymatic production of methyl ester from tributyrin. It was observed that 100% excess amount (1:6 mol ratio) of methanol/methyl acetate makes no negative effect on the enzyme activity. The initial reaction rate decreases as the tributyrin concentration increases. Consequently, tributyrin causes an inhibition at high concentrations. When the tributyrin concentration was used above the stoichiometric ratio (1:3 mol ratio), it decreased the activity of the enzyme. Michaelis-Menten parameters (K m) for tributyrin and methanol/methyl acetate were calculated as 0.1 and 50 M, respectively. An uncompetitive substrate inhibition constant for tributyrin was determined as 22.42 M. Experimental results were found to correlate well with the results of the kinetic model according to the ping-pong bi-bi mechanism. High conversions up to 90% were observed in a semicontinuous fluidized bed with low enzyme levels (1%). Conversions decreased with increasing flow rates.
Plants, algae, and cyanobacteria fix carbon dioxide to organic carbon with the Calvin-Benson (CB)... more Plants, algae, and cyanobacteria fix carbon dioxide to organic carbon with the Calvin-Benson (CB) cycle. Phosphoribulokinase (PRK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are essential CB-cycle enzymes that control substrate availability for the carboxylation enzyme Rubisco. PRK consumes ATP to produce the Rubisco substrate ribulose bisphosphate (RuBP). GAPDH catalyzes the reduction step of the CB cycle with NADPH to produce the sugar glyceraldehyde 3-phosphate (GAP), which is used for regeneration of RuBP and is the main exit point of the cycle. GAPDH and PRK are coregulated by the redox state of a conditionally disordered protein CP12, which forms a ternary complex with both enzymes. However, the structural basis of CB-cycle regulation by CP12 is unknown. Here, we show how CP12 modulates the activity of both GAPDH and PRK. Using thermophilic cyanobacte-rial homologs, we solve crystal structures of GAPDH with different cofactors and CP12 bound, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide preorders CP12 prior to binding the PRK active site, which is resolved in complex with CP12. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our structural and biochemical data explain how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation. redox regulation | carbon fixation | photosynthesis | Calvin-Benson cycle
The biological route for nitrogen gas entering the biosphere is reduction to ammonia by the nitro... more The biological route for nitrogen gas entering the biosphere is reduction to ammonia by the nitrogenase enzyme, which is inactivated by oxygen. Three types of nitrogenase exist, the least studied of which is the iron-only nitrogenase. The Anf3 protein in the bacterium Rhodobacter capsulatus is essential for diazotrophic (i.e. nitrogen-fixing) growth with the iron-only nitrogenase, but its enzymatic activity and function are unknown. Here, we biochemically and structurally characterize Anf3 from the model diazotrophic bacterium Azotobacter vinelandii. Determining the Anf3 crystal structure to atomic resolution, we observed that it is a dimeric flavocytochrome with an unusually close interaction between the heme and the flavin adenine dinucleotide cofactors. Measuring the reduction potentials by spectroelectrochemical redox titration, we observed values of -420 ± 10 mV and -330 ± 10 mV for the two FAD potentials and -340 ± 1 mV for the heme. We further show that Anf3 accepts electrons from spinach ferredoxin and that Anf3 consumes oxygen without generating superoxide or hydrogen peroxide. We predict that Anf3 protects the iron-only nitrogenase from oxygen inactivation by functioning as an oxidase in respiratory protection, with flavodoxin or ferredoxin as the physiological electron donors.
Microorganisms use carboxylase enzymes to form new carbon-carbon bonds by introducing carbon diox... more Microorganisms use carboxylase enzymes to form new carbon-carbon bonds by introducing carbon dioxide gas (CO 2) or its hydrated form, bicarbonate (HCO 3 −), into target molecules. Acetone carboxylases (ACs) catalyze the conversion of substrates acetone and HCO 3 − to form the product acetoacetate. Many bicarbonate-incorporating carboxylases rely on the organic cofactor biotin for the activation of bicarbonate. ACs contain metal ions but not organic cofactors, and use ATP to activate substrates through phosphorylation. How the enzyme coordinates these phosphorylation events and new CC bond formation in the absence of biotin has remained a mystery since these enzymes were discovered. The first structural rationale for acetone carboxylation is presented here, focusing on the 360 kDa (αβγ) 2 heterohexameric AC from Xanthobacter autotrophicus in the ligand-free, AMP-bound, and acetate coordinated states. These structures suggest successive steps in a catalytic cycle revealing that AC undergoes large conformational changes coupled to substrate activation by ATP to perform CC bond ligation at a distant Mn center. These results illustrate a new chemical strategy for the conversion of CO 2 into biomass, a process of great significance to the global carbon cycle. Carboxylases are enzymes that catalyze the incorporation of CO 2 into organic substrates. Assimilatory carboxy-lases use the carboxylation reaction to directly transform diverse carbon sources into central metabolites as part of autotrophic (photosynthetic) or heterotrophic metabolic pathways. The latter pathways are essential for the biological assimilation of often chemically intransigent, poorly activated organic compounds 1. Most enzymatic carboxylation reactions follow the same mechanistic principle: nucleophilic activation of substrates and electro-philic activation of CO 2 2. However, the stepwise mechanisms of carboxylation reactions differ in essential ways with respect to co-substrate, co-factor or metal requirements. Knowledge of these mechanisms provides the basis for an increased fundamental understanding of carboxylation chemistry, and contributes to future strategies for CO 2 capture. These in turn may mitigate the effects of increasing concentrations of CO 2 on the global climate 3. Acetone carboxylases are assimilatory carboxylases that catalyze the conversion of substrates acetone and HCO 3 − to form the product acetoacetate (Fig. 1a) allowing bacteria to incorporate this small, volatile and environmentally toxic ketone into biomass. In general, bicarbonate-dependent carboxylases catalyze the net dehydration of H 2 CO 3 , retaining CO 2 as a biotin adduct 4. However, ACs purified from multiple bacterial sources have been shown to be free of biotin or any other organic cofactor, instead containing quantities of manganese, zinc, and iron within a heteromultimeric protein complex 5–8. These carboxylases were also shown to convert ATP to AMP and two inorganic phosphate anions, suggesting that they catalyze the phosphorylation-dependent activation of both carbon substrates from a single nucleotide 9. This reaction sets ACs apart from the phylogenetically related acetophenone carboxylases (APCs). APC hydrolyses two ATP to ADP in order to activate acetophenone and bicarbonate 10. The AC β subunit and APC α subunits share homology and both possess nucleotide-binding sites. The structure of APC was recently determined , revealing that the two MgATP binding sites that are the proposed sites for the activation of acetophenone and bicarbonate are separated by ~50 Å. A large conformational shift was proposed to bring the two phosphoryl-ated intermediates in closer proximity for catalysis 11 .
The ability to design and construct structures with atomic level precision is one of the key goal... more The ability to design and construct structures with atomic level precision is one of the key goals of nanotechnology. Proteins offer an attractive target for atomic design because they can be synthesized chemically or biologically and can self-assemble. However, the generalized protein folding and design problem is unsolved. One approach to simplifying the problem is to use a repetitive protein as a scaffold. Repeat proteins are intrinsically modular, and their folding and structures are better understood than large globular domains. Here, we have developed a class of synthetic repeat proteins based on the pentapeptide repeat family of beta-solenoid proteins. We have constructed length variants of the basic scaffold and computa-tionally designed de novo loops projecting from the scaffold core. The experimentally solved 3.56-Å resolution crystal structure of one designed loop matches closely the designed hairpin structure, showing the computational design of a backbone extension onto a synthetic protein core without the use of backbone fragments from known structures. Two other loop designs were not clearly resolved in the crystal structures, and one loop appeared to be in an incorrect conformation. We have also shown that the repeat unit can accommodate whole-domain insertions by inserting a domain into one of the designed loops. computational protein design | synthetic repeat proteins | de novo backbone design | coarse-grained model D uring the course of evolution, natural proteins may be recruited to new unrelated functions conferring a selective advantage to the organism (1, 2). This accretion of new features and functions is likely to have left behind complex interlocking amino acid dependencies that can make reengineering natural proteins difficult and unpredictable (3). For this reason, we and others hypothesize that it is more desirable to design de novo proteins because these proteins provide a biologically neutral platform onto which functional elements can be grafted (4). Artificial proteins have been designed by decoding simple residue patterning rules that govern the packing of secondary structural elements, and this technique has been particularly successful for α-helical bundle proteins (5–7). An alternative approach is to assemble de novo folds from backbone fragments of known structures or idealized secondary structural elements and use computational protein design methods to design the sequence (4, 8–10). Both the computational and simpler rules-based design approaches have concentrated on designing proteins consisting of canonical secondary structure linked with loops of minimal length. A class of proteins that has attracted considerable interest is artificial proteins based on repeating structural motifs due to their intrinsic modularity and designability (11). Repeat proteins have applications that include their use as novel nanomaterials (12–14) and as scaffolds for molecular recognition (15, 16). These proteins may be designed using sequence consensus-based rules (17) or computational protein design methods (18, 19). There are a number of families of beta-helical repeat proteins (20), from which we chose the pentapeptide repeat family, forming the repeat five residues (RFR)-fold, which has a square cross-sectional profile, as the basis for the design of a class of synthetic repeat proteins (21) (Fig. 1 A and B). The RFR-fold has a number of properties that make it attractive as a substrate for design. The structure is unusually regular, but is able to tolerate a wide range of residues on the outside of the solenoid barrel. The solenoids in natural RFR-fold proteins are nearly straight in contrast to several other forms of repeat proteins, such as the leucine-rich repeat proteins, which are highly curved. There are examples of natural RFR-fold proteins with loop extensions projecting from the barrel, making this class of proteins particularly suitable for functionalization. The protein is similar in diameter to DNA, and some RFR-fold proteins are thought to play a role as DNA mimics (22). Here, we have designed and solved the structures of a number of artificial RFR-fold proteins of different lengths. Previously, computationally designed enzymes have reused backbone scaffolds from known natural proteins (23–25), although artificial helical bundle proteins have been functionalized using an intuitive manual design process (26–28). As the field of enzyme design becomes more ambitious, it is likely that consideration of backbone plasticity will become increasingly important (29). Backbone conformations from solved protein structures are guaranteed to be designable because there is at Significance The development of algorithms to design new proteins with backbone plasticity is a key challenge in computational protein design. In this paper, we describe a class of extensible synthetic repeat protein scaffolds with computationally designed variable loops projecting from the central core. We have developed methods to sample backbone conformations computationally using a coarse-grained potential energy function without using backbone fragments from known protein structures. This procedure was combined with existing methods for sequence design to successfully design a loop at atomic level precision. Given the inherent modular and composable nature of repeat proteins, this approach allows the iterative atomic-resolution design of complex structures with potential applications in novel nanomaterials and molecular recognition.
The dual-function fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) in cyanobacteria car... more The dual-function fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) in cyanobacteria carries out two activities in the Calvin cycle. Structures of this enzyme from the cyanobacterium Synechocystis sp. PCC 6803 exist, but only with adenosine monophosphate (AMP) or fructose-1,6-bisphosphate and AMP bound. The mechanisms which control both selectivity between the two sugars and the structural mechanisms for redox control are still unresolved. Here, the structure of the dual-function FBP/SBPase from the thermophilic cyanobacterium Thermosynechococcus elongatus is presented with sedoheptulose-7-phosphate bound and in the absence of AMP. The structure is globally very similar to the Synechocystis sp. PCC 6803 enzyme, but highlights features of selectivity at the active site and loop ordering at the AMP-binding site. Understanding the selectivity and control of this enzyme is critical for understanding the Calvin cycle in cyanobacteria and for possible biotechnological application in plants.
Phycocyanobilin:ferredoxinoxidoreductase (PcyA) catalyzes the proton-coupled four-electron reduct... more Phycocyanobilin:ferredoxinoxidoreductase (PcyA) catalyzes the proton-coupled four-electron reduction of biliverdin IXα's two vinyl groups to produce phycocyanobilin, an essential chromophore for phytochromes, cyanobacteriochromes and phycobiliproteins.Previous site directed mutagenesis studies indicated that the fully conserved residue His74 plays a critical role in the H-bonding network that permits proton transfer. Here, we exploit X-ray crystallography, enzymology, and molecular dynamics simulations to understand the functional role of this invariant histidine. The structures of the H74A, H74E, and H74Q variants of PcyA reveal that a "conserved" buried water molecule that bridges His74 and catalytically essential His88is not required for activity. Despite distinct conformations of Glu74 and Gln74 in theH74E and H74Q variants, both retain reasonable activity while the H74A variant is inactive, suggestingsmaller residues may generate cavities that increase flexibility, thereby reducing enzymatic activity. Molecular dynamic simulationsfurtherreveal that the crucial active site residue Asp105 is more dynamic in H74A compared towild-typePcyA and the two other His74 variants, supporting the conclusion that theAla74 mutation has increased the flexibility of the active site.
ABSTRACTHigh-resolution biomacromolecular structure determination is essential to better understa... more ABSTRACTHigh-resolution biomacromolecular structure determination is essential to better understand protein function and dynamics. Serial crystallography is an emerging structural biology technique which has fundamental limitations due to either sample volume requirements or immediate access to the competitive X-ray beamtime. Obtaining a high volume of well-diffracting, sufficient-size crystals while mitigating radiation damage remains a critical bottleneck of serial crystallography. As an alternative, we introduce the plate-reader module adapted for using a 72-well Terasaki plate for biomacromolecule structure determination at a convenience of a home X-ray source. We also present the first ambient temperature lysozyme structure determined at the Turkish Light Source (Turkish DeLight). The complete dataset was collected in 18.5 mins with resolution extending to 2.39 Å and 100% completeness. Combined with our previous cryogenic structure (PDB ID: 7Y6A), the ambient temperature struct...
X-ray crystallography is a robust and powerful structural biology technique that provides high-re... more X-ray crystallography is a robust and powerful structural biology technique that provides high-resolution atomic structures of biomacromolecules. Scientists use this technique to unravel mechanistic and structural details of biological macromolecules (e.g. proteins, nucleic acids, protein complexes, protein-nucleic acid complexes, or large biological compartments). Since its inception, single-crystal cryo-crystallography has never been performed in Türkiye due to the lack of a single-crystal X-ray diffractometer. The X-ray diffraction facility recently established at the University of Health Sciences, Istanbul, Türkiye will enable Turkish and international researchers to easily perform high-resolution structural analysis of biomacromolecules from single crystals. Here, we describe the technical and practical outlook of a state-of-the-art home-source X-ray, using lysozyme as a model protein. The methods and practice described in this article can be applied to any biological sample fo...
The biological route for nitrogen gas entering the biosphere is reduction to ammonia by the nitro... more The biological route for nitrogen gas entering the biosphere is reduction to ammonia by the nitrogenase enzyme, which is inactivated by oxygen. Three types of nitrogenase exist, the least studied of which is the iron-only nitrogenase. The Anf3 protein in the bacterium Rhodobacter capsulatus is essential for diazotrophic (i.e. nitrogen-fixing) growth with the iron-only nitrogenase, but its enzymatic activity and function are unknown. Here, we biochemically and structurally characterize Anf3 from the model diazotrophic bacterium Azotobacter vinelandii. Determining the Anf3 crystal structure to atomic resolution, we observed that it is a dimeric flavocytochrome with an unusually close interaction between the heme and the flavin adenine dinucleotide cofactors. Measuring the reduction potentials by spectroelectrochemical redox titration, we observed values of-420 ± 10 mV and-330 ± 10 mV for the two FAD potentials and-340 ± 1 mV for the heme. We further show that Anf3 accepts electrons from spinach ferredoxin and that Anf3 consumes oxygen without generating superoxide or hydrogen peroxide. We predict that Anf3 protects the iron-only nitrogenase from oxygen inactivation by functioning as an oxidase in respiratory protection, with flavodoxin or ferredoxin as the physiological electron donors.
Microorganisms use carboxylase enzymes to form new carbon-carbon bonds by introducing carbon diox... more Microorganisms use carboxylase enzymes to form new carbon-carbon bonds by introducing carbon dioxide gas (CO2) or its hydrated form, bicarbonate (HCO3(-)), into target molecules. Acetone carboxylases (ACs) catalyze the conversion of substrates acetone and HCO3(-) to form the product acetoacetate. Many bicarbonate-incorporating carboxylases rely on the organic cofactor biotin for the activation of bicarbonate. ACs contain metal ions but not organic cofactors, and use ATP to activate substrates through phosphorylation. How the enzyme coordinates these phosphorylation events and new C-C bond formation in the absence of biotin has remained a mystery since these enzymes were discovered. The first structural rationale for acetone carboxylation is presented here, focusing on the 360 kDa (αβγ)2 heterohexameric AC from Xanthobacter autotrophicus in the ligand-free, AMP-bound, and acetate coordinated states. These structures suggest successive steps in a catalytic cycle revealing that AC unde...
The dual-function fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) in cyanobacteria car... more The dual-function fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) in cyanobacteria carries out two activities in the Calvin cycle. Structures of this enzyme from the cyanobacterium Synechocystis sp. PCC 6803 exist, but only with adenosine monophosphate (AMP) or fructose-1,6-bisphosphate and AMP bound. The mechanisms which control both selectivity between the two sugars and the structural mechanisms for redox control are still unresolved. Here, the structure of the dual-function FBP/SBPase from the thermophilic cyanobacterium Thermosynechococcus elongatus is presented with sedoheptulose-7-phosphate bound and in the absence of AMP. The structure is globally very similar to the Synechocystis sp. PCC 6803 enzyme, but highlights features of selectivity at the active site and loop ordering at the AMP-binding site. Understanding the selectivity and control of this enzyme is critical for understanding the Calvin cycle in cyanobacteria and for possible biotechnological application...
The goal of this study was to develop and validate a liquid chromatography (LC) method for the ra... more The goal of this study was to develop and validate a liquid chromatography (LC) method for the rapid determination of dipyrone (metamizole sodium) in solid and liquid formulations. Samples were extracted with water and filtered prior to LC analysis. The analytical procedure involved reversed phase LC with a Zorbax SB C 18 column (5 m particle size, 4.6 mm × 250 mm i.d.), isoctratic methanol/water (80/20, v/v) mobile phase with UV detection at 254 nm. The flow rate was 1 mL min -1 , injection volume was 100 L and retention time of dipyrone was 3.3 min. The method was validated with respect to linearity, precision and accuracy. Calibration curves were linear in the range 0.5-100 g mL -1 for analyte and the recoveries at three levels (10, 15 and 25 g mL -1 ) ranged from 93 to 100%. All results for dipyrone were confirmed by liquid chromatography-mass spectrometry (LC-MS) and 1 H nuclear magnetic resonance (NMR) spectroscopy. Due to its simplicity and accuracy, the assay method is suitable for routine analysis of both solid and liquid formulations and seven retail samples were analyzed using the validated method.
In plants, carbon dioxide is fixed via the Calvin cycle in a tightly regulated process. Key to th... more In plants, carbon dioxide is fixed via the Calvin cycle in a tightly regulated process. Key to this regulation is the conditionally disordered protein CP12. CP12 forms a complex with two Calvin cycle enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), inhibiting their activities. The mode of CP12 action was unknown. By solving crystal structures of CP12 bound to GAPDH, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide pre-orders CP12 prior to binding the PRK active site. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our model explains how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation.One Sentence SummaryHow plants turn off carbon fixation in the dark.
Transesterification by methanol and interesterification by methyl acetate were performed simultan... more Transesterification by methanol and interesterification by methyl acetate were performed simultaneously, and the advantages of using methyl acetate with methanol were investigated in the enzymatic production of methyl ester from tributyrin. It was observed that 100% excess amount (1:6 mol ratio) of methanol/methyl acetate makes no negative effect on the enzyme activity. The initial reaction rate decreases as the tributyrin concentration increases. Consequently, tributyrin causes an inhibition at high concentrations. When the tributyrin concentration was used above the stoichiometric ratio (1:3 mol ratio), it decreased the activity of the enzyme. Michaelis-Menten parameters (K m) for tributyrin and methanol/methyl acetate were calculated as 0.1 and 50 M, respectively. An uncompetitive substrate inhibition constant for tributyrin was determined as 22.42 M. Experimental results were found to correlate well with the results of the kinetic model according to the ping-pong bi-bi mechanism. High conversions up to 90% were observed in a semicontinuous fluidized bed with low enzyme levels (1%). Conversions decreased with increasing flow rates.
Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the proton-coupled four-electron reduc... more Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the proton-coupled four-electron reduction 27 of biliverdin IXa's two vinyl groups to produce phycocyanobilin, an essential chromophore for phyto-28 chromes, cyanobacteriochromes and phycobiliproteins. Previous site directed mutagenesis studies indi-29 cated that the fully conserved residue His74 plays a critical role in the H-bonding network that permits 30 proton transfer. Here, we exploit X-ray crystallography, enzymology and molecular dynamics simulations 31 to understand the functional role of this invariant histidine. The structures of the H74A, H74E and H74Q 32 variants of PcyA reveal that a ''conserved'' buried water molecule that bridges His74 and catalytically 33 essential His88 is not required for activity. Despite distinct conformations of Glu74 and Gln74 in theH74E 34 and H74Q variants, both retain reasonable activity while the H74A variant is inactive, suggesting smaller 35 residues may generate cavities that increase flexibility, thereby reducing enzymatic activity. Molecular 36 dynamic simulations further reveal that the crucial active site residue Asp105 is more dynamic in 37 H74A compared to wild-type PcyA and the two other His74 variants, supporting the conclusion that 38 the Ala74 mutation has increased the flexibility of the active site.
Proceedings of the National Academy of Sciences of the United States of America, Sep 13, 2016
The ability to design and construct structures with atomic level precision is one of the key goal... more The ability to design and construct structures with atomic level precision is one of the key goals of nanotechnology. Proteins offer an attractive target for atomic design because they can be synthesized chemically or biologically and can self-assemble. However, the generalized protein folding and design problem is unsolved. One approach to simplifying the problem is to use a repetitive protein as a scaffold. Repeat proteins are intrinsically modular, and their folding and structures are better understood than large globular domains. Here, we have developed a class of synthetic repeat proteins based on the pentapeptide repeat family of beta-solenoid proteins. We have constructed length variants of the basic scaffold and computationally designed de novo loops projecting from the scaffold core. The experimentally solved 3.56-Å resolution crystal structure of one designed loop matches closely the designed hairpin structure, showing the computational design of a backbone extension onto ...
Transesterification by methanol and interesterification by methyl acetate were performed simultan... more Transesterification by methanol and interesterification by methyl acetate were performed simultaneously, and the advantages of using methyl acetate with methanol were investigated in the enzymatic production of methyl ester from tributyrin. It was observed that 100% excess amount (1:6 mol ratio) of methanol/methyl acetate makes no negative effect on the enzyme activity. The initial reaction rate decreases as the tributyrin concentration increases. Consequently, tributyrin causes an inhibition at high concentrations. When the tributyrin concentration was used above the stoichiometric ratio (1:3 mol ratio), it decreased the activity of the enzyme. Michaelis-Menten parameters (K m) for tributyrin and methanol/methyl acetate were calculated as 0.1 and 50 M, respectively. An uncompetitive substrate inhibition constant for tributyrin was determined as 22.42 M. Experimental results were found to correlate well with the results of the kinetic model according to the ping-pong bi-bi mechanism. High conversions up to 90% were observed in a semicontinuous fluidized bed with low enzyme levels (1%). Conversions decreased with increasing flow rates.
Plants, algae, and cyanobacteria fix carbon dioxide to organic carbon with the Calvin-Benson (CB)... more Plants, algae, and cyanobacteria fix carbon dioxide to organic carbon with the Calvin-Benson (CB) cycle. Phosphoribulokinase (PRK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are essential CB-cycle enzymes that control substrate availability for the carboxylation enzyme Rubisco. PRK consumes ATP to produce the Rubisco substrate ribulose bisphosphate (RuBP). GAPDH catalyzes the reduction step of the CB cycle with NADPH to produce the sugar glyceraldehyde 3-phosphate (GAP), which is used for regeneration of RuBP and is the main exit point of the cycle. GAPDH and PRK are coregulated by the redox state of a conditionally disordered protein CP12, which forms a ternary complex with both enzymes. However, the structural basis of CB-cycle regulation by CP12 is unknown. Here, we show how CP12 modulates the activity of both GAPDH and PRK. Using thermophilic cyanobacte-rial homologs, we solve crystal structures of GAPDH with different cofactors and CP12 bound, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide preorders CP12 prior to binding the PRK active site, which is resolved in complex with CP12. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our structural and biochemical data explain how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation. redox regulation | carbon fixation | photosynthesis | Calvin-Benson cycle
The biological route for nitrogen gas entering the biosphere is reduction to ammonia by the nitro... more The biological route for nitrogen gas entering the biosphere is reduction to ammonia by the nitrogenase enzyme, which is inactivated by oxygen. Three types of nitrogenase exist, the least studied of which is the iron-only nitrogenase. The Anf3 protein in the bacterium Rhodobacter capsulatus is essential for diazotrophic (i.e. nitrogen-fixing) growth with the iron-only nitrogenase, but its enzymatic activity and function are unknown. Here, we biochemically and structurally characterize Anf3 from the model diazotrophic bacterium Azotobacter vinelandii. Determining the Anf3 crystal structure to atomic resolution, we observed that it is a dimeric flavocytochrome with an unusually close interaction between the heme and the flavin adenine dinucleotide cofactors. Measuring the reduction potentials by spectroelectrochemical redox titration, we observed values of -420 ± 10 mV and -330 ± 10 mV for the two FAD potentials and -340 ± 1 mV for the heme. We further show that Anf3 accepts electrons from spinach ferredoxin and that Anf3 consumes oxygen without generating superoxide or hydrogen peroxide. We predict that Anf3 protects the iron-only nitrogenase from oxygen inactivation by functioning as an oxidase in respiratory protection, with flavodoxin or ferredoxin as the physiological electron donors.
Microorganisms use carboxylase enzymes to form new carbon-carbon bonds by introducing carbon diox... more Microorganisms use carboxylase enzymes to form new carbon-carbon bonds by introducing carbon dioxide gas (CO 2) or its hydrated form, bicarbonate (HCO 3 −), into target molecules. Acetone carboxylases (ACs) catalyze the conversion of substrates acetone and HCO 3 − to form the product acetoacetate. Many bicarbonate-incorporating carboxylases rely on the organic cofactor biotin for the activation of bicarbonate. ACs contain metal ions but not organic cofactors, and use ATP to activate substrates through phosphorylation. How the enzyme coordinates these phosphorylation events and new CC bond formation in the absence of biotin has remained a mystery since these enzymes were discovered. The first structural rationale for acetone carboxylation is presented here, focusing on the 360 kDa (αβγ) 2 heterohexameric AC from Xanthobacter autotrophicus in the ligand-free, AMP-bound, and acetate coordinated states. These structures suggest successive steps in a catalytic cycle revealing that AC undergoes large conformational changes coupled to substrate activation by ATP to perform CC bond ligation at a distant Mn center. These results illustrate a new chemical strategy for the conversion of CO 2 into biomass, a process of great significance to the global carbon cycle. Carboxylases are enzymes that catalyze the incorporation of CO 2 into organic substrates. Assimilatory carboxy-lases use the carboxylation reaction to directly transform diverse carbon sources into central metabolites as part of autotrophic (photosynthetic) or heterotrophic metabolic pathways. The latter pathways are essential for the biological assimilation of often chemically intransigent, poorly activated organic compounds 1. Most enzymatic carboxylation reactions follow the same mechanistic principle: nucleophilic activation of substrates and electro-philic activation of CO 2 2. However, the stepwise mechanisms of carboxylation reactions differ in essential ways with respect to co-substrate, co-factor or metal requirements. Knowledge of these mechanisms provides the basis for an increased fundamental understanding of carboxylation chemistry, and contributes to future strategies for CO 2 capture. These in turn may mitigate the effects of increasing concentrations of CO 2 on the global climate 3. Acetone carboxylases are assimilatory carboxylases that catalyze the conversion of substrates acetone and HCO 3 − to form the product acetoacetate (Fig. 1a) allowing bacteria to incorporate this small, volatile and environmentally toxic ketone into biomass. In general, bicarbonate-dependent carboxylases catalyze the net dehydration of H 2 CO 3 , retaining CO 2 as a biotin adduct 4. However, ACs purified from multiple bacterial sources have been shown to be free of biotin or any other organic cofactor, instead containing quantities of manganese, zinc, and iron within a heteromultimeric protein complex 5–8. These carboxylases were also shown to convert ATP to AMP and two inorganic phosphate anions, suggesting that they catalyze the phosphorylation-dependent activation of both carbon substrates from a single nucleotide 9. This reaction sets ACs apart from the phylogenetically related acetophenone carboxylases (APCs). APC hydrolyses two ATP to ADP in order to activate acetophenone and bicarbonate 10. The AC β subunit and APC α subunits share homology and both possess nucleotide-binding sites. The structure of APC was recently determined , revealing that the two MgATP binding sites that are the proposed sites for the activation of acetophenone and bicarbonate are separated by ~50 Å. A large conformational shift was proposed to bring the two phosphoryl-ated intermediates in closer proximity for catalysis 11 .
The ability to design and construct structures with atomic level precision is one of the key goal... more The ability to design and construct structures with atomic level precision is one of the key goals of nanotechnology. Proteins offer an attractive target for atomic design because they can be synthesized chemically or biologically and can self-assemble. However, the generalized protein folding and design problem is unsolved. One approach to simplifying the problem is to use a repetitive protein as a scaffold. Repeat proteins are intrinsically modular, and their folding and structures are better understood than large globular domains. Here, we have developed a class of synthetic repeat proteins based on the pentapeptide repeat family of beta-solenoid proteins. We have constructed length variants of the basic scaffold and computa-tionally designed de novo loops projecting from the scaffold core. The experimentally solved 3.56-Å resolution crystal structure of one designed loop matches closely the designed hairpin structure, showing the computational design of a backbone extension onto a synthetic protein core without the use of backbone fragments from known structures. Two other loop designs were not clearly resolved in the crystal structures, and one loop appeared to be in an incorrect conformation. We have also shown that the repeat unit can accommodate whole-domain insertions by inserting a domain into one of the designed loops. computational protein design | synthetic repeat proteins | de novo backbone design | coarse-grained model D uring the course of evolution, natural proteins may be recruited to new unrelated functions conferring a selective advantage to the organism (1, 2). This accretion of new features and functions is likely to have left behind complex interlocking amino acid dependencies that can make reengineering natural proteins difficult and unpredictable (3). For this reason, we and others hypothesize that it is more desirable to design de novo proteins because these proteins provide a biologically neutral platform onto which functional elements can be grafted (4). Artificial proteins have been designed by decoding simple residue patterning rules that govern the packing of secondary structural elements, and this technique has been particularly successful for α-helical bundle proteins (5–7). An alternative approach is to assemble de novo folds from backbone fragments of known structures or idealized secondary structural elements and use computational protein design methods to design the sequence (4, 8–10). Both the computational and simpler rules-based design approaches have concentrated on designing proteins consisting of canonical secondary structure linked with loops of minimal length. A class of proteins that has attracted considerable interest is artificial proteins based on repeating structural motifs due to their intrinsic modularity and designability (11). Repeat proteins have applications that include their use as novel nanomaterials (12–14) and as scaffolds for molecular recognition (15, 16). These proteins may be designed using sequence consensus-based rules (17) or computational protein design methods (18, 19). There are a number of families of beta-helical repeat proteins (20), from which we chose the pentapeptide repeat family, forming the repeat five residues (RFR)-fold, which has a square cross-sectional profile, as the basis for the design of a class of synthetic repeat proteins (21) (Fig. 1 A and B). The RFR-fold has a number of properties that make it attractive as a substrate for design. The structure is unusually regular, but is able to tolerate a wide range of residues on the outside of the solenoid barrel. The solenoids in natural RFR-fold proteins are nearly straight in contrast to several other forms of repeat proteins, such as the leucine-rich repeat proteins, which are highly curved. There are examples of natural RFR-fold proteins with loop extensions projecting from the barrel, making this class of proteins particularly suitable for functionalization. The protein is similar in diameter to DNA, and some RFR-fold proteins are thought to play a role as DNA mimics (22). Here, we have designed and solved the structures of a number of artificial RFR-fold proteins of different lengths. Previously, computationally designed enzymes have reused backbone scaffolds from known natural proteins (23–25), although artificial helical bundle proteins have been functionalized using an intuitive manual design process (26–28). As the field of enzyme design becomes more ambitious, it is likely that consideration of backbone plasticity will become increasingly important (29). Backbone conformations from solved protein structures are guaranteed to be designable because there is at Significance The development of algorithms to design new proteins with backbone plasticity is a key challenge in computational protein design. In this paper, we describe a class of extensible synthetic repeat protein scaffolds with computationally designed variable loops projecting from the central core. We have developed methods to sample backbone conformations computationally using a coarse-grained potential energy function without using backbone fragments from known protein structures. This procedure was combined with existing methods for sequence design to successfully design a loop at atomic level precision. Given the inherent modular and composable nature of repeat proteins, this approach allows the iterative atomic-resolution design of complex structures with potential applications in novel nanomaterials and molecular recognition.
The dual-function fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) in cyanobacteria car... more The dual-function fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) in cyanobacteria carries out two activities in the Calvin cycle. Structures of this enzyme from the cyanobacterium Synechocystis sp. PCC 6803 exist, but only with adenosine monophosphate (AMP) or fructose-1,6-bisphosphate and AMP bound. The mechanisms which control both selectivity between the two sugars and the structural mechanisms for redox control are still unresolved. Here, the structure of the dual-function FBP/SBPase from the thermophilic cyanobacterium Thermosynechococcus elongatus is presented with sedoheptulose-7-phosphate bound and in the absence of AMP. The structure is globally very similar to the Synechocystis sp. PCC 6803 enzyme, but highlights features of selectivity at the active site and loop ordering at the AMP-binding site. Understanding the selectivity and control of this enzyme is critical for understanding the Calvin cycle in cyanobacteria and for possible biotechnological application in plants.
Phycocyanobilin:ferredoxinoxidoreductase (PcyA) catalyzes the proton-coupled four-electron reduct... more Phycocyanobilin:ferredoxinoxidoreductase (PcyA) catalyzes the proton-coupled four-electron reduction of biliverdin IXα's two vinyl groups to produce phycocyanobilin, an essential chromophore for phytochromes, cyanobacteriochromes and phycobiliproteins.Previous site directed mutagenesis studies indicated that the fully conserved residue His74 plays a critical role in the H-bonding network that permits proton transfer. Here, we exploit X-ray crystallography, enzymology, and molecular dynamics simulations to understand the functional role of this invariant histidine. The structures of the H74A, H74E, and H74Q variants of PcyA reveal that a "conserved" buried water molecule that bridges His74 and catalytically essential His88is not required for activity. Despite distinct conformations of Glu74 and Gln74 in theH74E and H74Q variants, both retain reasonable activity while the H74A variant is inactive, suggestingsmaller residues may generate cavities that increase flexibility, thereby reducing enzymatic activity. Molecular dynamic simulationsfurtherreveal that the crucial active site residue Asp105 is more dynamic in H74A compared towild-typePcyA and the two other His74 variants, supporting the conclusion that theAla74 mutation has increased the flexibility of the active site.
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Papers by Burak Veli Kabasakal
Synechocystis
sp. PCC 6803 exist, but only with adenosine monophosphate (AMP) or fructose-1,6-bisphosphate and AMP bound. The mechanisms which control both selectivity between the two sugars and the structural mechanisms for redox control are still unresolved. Here, the structure of the dual-function FBP/SBPase from the thermophilic cyanobacterium
Thermosynechococcus elongatus
is presented with sedoheptulose-7-phosphate bound and in the absence of AMP. The structure is globally very similar to the
Synechocystis
sp. PCC 6803 enzyme, but highlights features of selectivity at the active site and loop ordering at the AMP-binding site. Understanding the selectivity and control of this enzyme is critical for understanding the Calvin cycle in cyanobacteria and for possible biotechnological application in plants.
Synechocystis
sp. PCC 6803 exist, but only with adenosine monophosphate (AMP) or fructose-1,6-bisphosphate and AMP bound. The mechanisms which control both selectivity between the two sugars and the structural mechanisms for redox control are still unresolved. Here, the structure of the dual-function FBP/SBPase from the thermophilic cyanobacterium
Thermosynechococcus elongatus
is presented with sedoheptulose-7-phosphate bound and in the absence of AMP. The structure is globally very similar to the
Synechocystis
sp. PCC 6803 enzyme, but highlights features of selectivity at the active site and loop ordering at the AMP-binding site. Understanding the selectivity and control of this enzyme is critical for understanding the Calvin cycle in cyanobacteria and for possible biotechnological application in plants.