Heme pocket structural properties of a bacterial truncated hemoglobin from thermobifida fusca
An acidic surface variant (ASV) of the "truncated" hemoglobin from Thermobifida fusca was designed with the aim of creating a versatile globin scaffold endowed with thermostability and a high level of recombinant expression in its soluble form while keeping the active site unmodified. This...
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2010
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00062960_v49_n49_p10394_Droghetti http://hdl.handle.net/20.500.12110/paper_00062960_v49_n49_p10394_Droghetti |
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paper:paper_00062960_v49_n49_p10394_Droghetti2023-06-08T14:30:46Z Heme pocket structural properties of a bacterial truncated hemoglobin from thermobifida fusca Acidic surface Active site Co complexes Computational results Conserved polar residues Heme pockets Molecular dynamics simulations Protein structures Recombinant expression Resonance Raman spectroscopy Stretching frequency Thermobifida fusca Truncated hemoglobins Wild types Wild-type proteins Hemoglobin Porphyrins Raman spectroscopy Molecular dynamics arginine carbon monoxide glutamic acid heme histidine iron phenylalanine truncated hemoglobin tyrosine acidic surface variant article bacterium hydrogen bond infrared spectroscopy molecular dynamics mutagenesis nonhuman priority journal protein structure Raman spectrometry simulation Thermobifida fusca thermostability Actinomycetales Bacterial Proteins Heme Mutation Protein Binding Recombinant Proteins Truncated Hemoglobins Bacteria (microorganisms) Thermobifida fusca An acidic surface variant (ASV) of the "truncated" hemoglobin from Thermobifida fusca was designed with the aim of creating a versatile globin scaffold endowed with thermostability and a high level of recombinant expression in its soluble form while keeping the active site unmodified. This engineered protein was obtained by mutating the surface-exposed residues Phe107 and Arg91 to Glu. Molecular dynamics simulations showed that the mutated residues remain solvent-exposed, not affecting the overall protein structure. Thus, the ASV was used in a combinatorial mutagenesis of the distal heme pocket residues in which one, two, or three of the conserved polar residues [TyrB10(54), TyrCD1(67), and TrpG8(119)] were substituted with Phe. Mutants were characterized by infrared and resonance Raman spectroscopy and compared with the wild-type protein. Similar Fe-proximal His stretching frequencies suggest that none of the mutations alters the proximal side of the heme cavity. Two conformers were observed in the spectra of the CO complexes of both wild-type and ASV protein: form 1 with v(FeC) and v(CO) at 509 and 1938 cm-1 and form 2 with v(FeC) and v(CO) at 518 and 1920 cm-1, respectively. Molecular dynamics simulations were performed for the wild-type and ASV forms, as well as for the TyrB10 mutant. The spectroscopic and computational results demonstrate that CO interacts with TrpG8 in form 1 and interacts with both TrpG8 and TyrCD1 in form 2. TyrB10 does not directly interact with the bound CO. © 2010 American Chemical Society. 2010 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00062960_v49_n49_p10394_Droghetti http://hdl.handle.net/20.500.12110/paper_00062960_v49_n49_p10394_Droghetti |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
Acidic surface Active site Co complexes Computational results Conserved polar residues Heme pockets Molecular dynamics simulations Protein structures Recombinant expression Resonance Raman spectroscopy Stretching frequency Thermobifida fusca Truncated hemoglobins Wild types Wild-type proteins Hemoglobin Porphyrins Raman spectroscopy Molecular dynamics arginine carbon monoxide glutamic acid heme histidine iron phenylalanine truncated hemoglobin tyrosine acidic surface variant article bacterium hydrogen bond infrared spectroscopy molecular dynamics mutagenesis nonhuman priority journal protein structure Raman spectrometry simulation Thermobifida fusca thermostability Actinomycetales Bacterial Proteins Heme Mutation Protein Binding Recombinant Proteins Truncated Hemoglobins Bacteria (microorganisms) Thermobifida fusca |
| spellingShingle |
Acidic surface Active site Co complexes Computational results Conserved polar residues Heme pockets Molecular dynamics simulations Protein structures Recombinant expression Resonance Raman spectroscopy Stretching frequency Thermobifida fusca Truncated hemoglobins Wild types Wild-type proteins Hemoglobin Porphyrins Raman spectroscopy Molecular dynamics arginine carbon monoxide glutamic acid heme histidine iron phenylalanine truncated hemoglobin tyrosine acidic surface variant article bacterium hydrogen bond infrared spectroscopy molecular dynamics mutagenesis nonhuman priority journal protein structure Raman spectrometry simulation Thermobifida fusca thermostability Actinomycetales Bacterial Proteins Heme Mutation Protein Binding Recombinant Proteins Truncated Hemoglobins Bacteria (microorganisms) Thermobifida fusca Heme pocket structural properties of a bacterial truncated hemoglobin from thermobifida fusca |
| topic_facet |
Acidic surface Active site Co complexes Computational results Conserved polar residues Heme pockets Molecular dynamics simulations Protein structures Recombinant expression Resonance Raman spectroscopy Stretching frequency Thermobifida fusca Truncated hemoglobins Wild types Wild-type proteins Hemoglobin Porphyrins Raman spectroscopy Molecular dynamics arginine carbon monoxide glutamic acid heme histidine iron phenylalanine truncated hemoglobin tyrosine acidic surface variant article bacterium hydrogen bond infrared spectroscopy molecular dynamics mutagenesis nonhuman priority journal protein structure Raman spectrometry simulation Thermobifida fusca thermostability Actinomycetales Bacterial Proteins Heme Mutation Protein Binding Recombinant Proteins Truncated Hemoglobins Bacteria (microorganisms) Thermobifida fusca |
| description |
An acidic surface variant (ASV) of the "truncated" hemoglobin from Thermobifida fusca was designed with the aim of creating a versatile globin scaffold endowed with thermostability and a high level of recombinant expression in its soluble form while keeping the active site unmodified. This engineered protein was obtained by mutating the surface-exposed residues Phe107 and Arg91 to Glu. Molecular dynamics simulations showed that the mutated residues remain solvent-exposed, not affecting the overall protein structure. Thus, the ASV was used in a combinatorial mutagenesis of the distal heme pocket residues in which one, two, or three of the conserved polar residues [TyrB10(54), TyrCD1(67), and TrpG8(119)] were substituted with Phe. Mutants were characterized by infrared and resonance Raman spectroscopy and compared with the wild-type protein. Similar Fe-proximal His stretching frequencies suggest that none of the mutations alters the proximal side of the heme cavity. Two conformers were observed in the spectra of the CO complexes of both wild-type and ASV protein: form 1 with v(FeC) and v(CO) at 509 and 1938 cm-1 and form 2 with v(FeC) and v(CO) at 518 and 1920 cm-1, respectively. Molecular dynamics simulations were performed for the wild-type and ASV forms, as well as for the TyrB10 mutant. The spectroscopic and computational results demonstrate that CO interacts with TrpG8 in form 1 and interacts with both TrpG8 and TyrCD1 in form 2. TyrB10 does not directly interact with the bound CO. © 2010 American Chemical Society. |
| title |
Heme pocket structural properties of a bacterial truncated hemoglobin from thermobifida fusca |
| title_short |
Heme pocket structural properties of a bacterial truncated hemoglobin from thermobifida fusca |
| title_full |
Heme pocket structural properties of a bacterial truncated hemoglobin from thermobifida fusca |
| title_fullStr |
Heme pocket structural properties of a bacterial truncated hemoglobin from thermobifida fusca |
| title_full_unstemmed |
Heme pocket structural properties of a bacterial truncated hemoglobin from thermobifida fusca |
| title_sort |
heme pocket structural properties of a bacterial truncated hemoglobin from thermobifida fusca |
| publishDate |
2010 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00062960_v49_n49_p10394_Droghetti http://hdl.handle.net/20.500.12110/paper_00062960_v49_n49_p10394_Droghetti |
| _version_ |
1768541874261327872 |