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Philip N Bryan - One of the best experts on this subject based on the ideXlab platform.
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subdomain interactions foster the design of two protein pairs with 80 Sequence Identity but different folds
Biophysical Journal, 2015Co-Authors: Lauren L Porter, Yihong Chen, John Orban, Philip N BryanAbstract:Metamorphic proteins, including proteins with high levels of Sequence Identity but different folds, are exceptions to the long-standing rule-of-thumb that proteins with as little as 30% Sequence Identity adopt the same fold. Which topologies can be bridged by these highly identical Sequences remains an open question. Here we bridge two 3-α-helix bundle proteins with two radically different folds. Using a straightforward approach, we engineered the Sequences of one subdomain within maltose binding protein (MBP, α/β/α-sandwich) and another within outer surface protein A (OspA, β-sheet) to have high Sequence Identity (80 and 77%, respectively) with engineered variants of protein G (GA, 3-α-helix bundle). Circular dichroism and nuclear magnetic resonance spectra of all engineered variants demonstrate that they maintain their native conformations despite substantial Sequence modification. Furthermore, the MBP variant (80% identical to GA) remained active. Thermodynamic analysis of numerous GA and MBP variants suggests that the key to our approach involved stabilizing the modified MBP and OspA subdomains via external interactions with neighboring substructures, indicating that subdomain interactions can stabilize alternative folds over a broad range of Sequence variation. These findings suggest that it is possible to bridge one fold with many other topologies, which has implications for protein folding, evolution, and misfolding diseases.
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Subdomain interactions foster the design of two protein pairs with ∼80% Sequence Identity but different folds.
Biophysical journal, 2015Co-Authors: Lauren L Porter, Yihong Chen, John Orban, Philip N BryanAbstract:Metamorphic proteins, including proteins with high levels of Sequence Identity but different folds, are exceptions to the long-standing rule-of-thumb that proteins with as little as 30% Sequence Identity adopt the same fold. Which topologies can be bridged by these highly identical Sequences remains an open question. Here we bridge two 3-α-helix bundle proteins with two radically different folds. Using a straightforward approach, we engineered the Sequences of one subdomain within maltose binding protein (MBP, α/β/α-sandwich) and another within outer surface protein A (OspA, β-sheet) to have high Sequence Identity (80 and 77%, respectively) with engineered variants of protein G (GA, 3-α-helix bundle). Circular dichroism and nuclear magnetic resonance spectra of all engineered variants demonstrate that they maintain their native conformations despite substantial Sequence modification. Furthermore, the MBP variant (80% identical to GA) remained active. Thermodynamic analysis of numerous GA and MBP variants suggests that the key to our approach involved stabilizing the modified MBP and OspA subdomains via external interactions with neighboring substructures, indicating that subdomain interactions can stabilize alternative folds over a broad range of Sequence variation. These findings suggest that it is possible to bridge one fold with many other topologies, which has implications for protein folding, evolution, and misfolding diseases.
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de novo structure generation using chemical shifts for proteins with high Sequence Identity but different folds
Protein Science, 2010Co-Authors: Yang Shen, Philip N Bryan, John Orban, David Baker, Ad BaxAbstract:Proteins with high-Sequence Identity but very different folds present a special challenge to Sequence-based protein structure prediction methods. In particular, a 56-residue three-helical bundle protein (GA95) and an α/β-fold protein (GB95), which share 95% Sequence Identity, were targets in the CASP-8 structure prediction contest. With only 12 out of 300 submitted server-CASP8 models for GA95 exhibiting the correct fold, this protein proved particularly challenging despite its small size. Here, we demonstrate that the information contained in NMR chemical shifts can readily be exploited by the CS-Rosetta structure prediction program and yields adequate convergence, even when input chemical shifts are limited to just amide 1HN and 15N or 1HN and 1Hα values.
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nmr structures of two designed proteins with high Sequence Identity but different fold and function
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Yihong Chen, Patrick Alexander, Philip N Bryan, John OrbanAbstract:How protein Sequence codes for 3D structure remains a fundamental question in biology. One approach to understanding the folding code is to design a pair of proteins with maximal Sequence Identity but retaining different folds. Therefore, the nonidentities must be responsible for determining which fold topology prevails and constitute a fold-specific folding code. We recently designed two proteins, GA88 and GB88, with 88% Sequence Identity but different folds and functions [Alexander et al. (2007) Proc Natl Acad Sci USA 104:11963–11968]. Here, we describe the detailed 3D structures of these proteins determined in solution by NMR spectroscopy. Despite a large number of mutations taking the Sequence Identity level from 16 to 88%, GA88 and GB88 maintain their distinct wild-type 3-α and α/β folds, respectively. To our knowledge, the 3D-structure determination of two monomeric proteins with such high Sequence Identity but different fold topology is unprecedented. The geometries of the seven nonidentical residues (of 56 total) provide insights into the structural basis for switching between 3-α and α/β conformations. Further mutation of a subset of these nonidentities, guided by the GA88 and GB88 structures, leads to proteins with even higher levels of Sequence Identity (95%) and different folds. Thus, conformational switching to an alternative monomeric fold of comparable stability can be effected with just a handful of mutations in a small protein. This result has implications for understanding not only the folding code but also the evolution of new folds.
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the design and characterization of two proteins with 88 Sequence Identity but different structure and function
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Patrick Alexander, Yihong Chen, John Orban, Philip N BryanAbstract:To identify a simplified code for conformational switching, we have redesigned two natural proteins to have 88% Sequence Identity but different tertiary structures: a 3-α helix fold and an α/β fold. We describe the design of these homologous heteromorphic proteins, their structural properties as determined by NMR, their conformational stabilities, and their affinities for their respective ligands: IgG and serum albumin. Each of these proteins is completely folded at 25°C, is monomeric, and retains the native binding activity. The complete binding epitope for both ligands is encoded within each of the proteins. The IgG-binding epitope is functional only in the α/β fold, and the albumin-binding epitope is functional only in the 3-α fold. These results demonstrate that two monomeric folds and two different functions can be encoded with only 12% of the amino acids in a protein (7 of 56). The fact that 49 aa in these proteins are compatible with both folds shows that the essential information determining a fold can be highly concentrated in a few amino acids and that a very limited subset of interactions in the protein can tip the balance from one monomer fold to another. This delicate balance helps explain why protein structure prediction is so challenging. Furthermore, because a few mutations can result in both new conformation and new function, the evolution of new folds driven by natural selection for alternative functions may be much more probable than previously recognized.
John Orban - One of the best experts on this subject based on the ideXlab platform.
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subdomain interactions foster the design of two protein pairs with 80 Sequence Identity but different folds
Biophysical Journal, 2015Co-Authors: Lauren L Porter, Yihong Chen, John Orban, Philip N BryanAbstract:Metamorphic proteins, including proteins with high levels of Sequence Identity but different folds, are exceptions to the long-standing rule-of-thumb that proteins with as little as 30% Sequence Identity adopt the same fold. Which topologies can be bridged by these highly identical Sequences remains an open question. Here we bridge two 3-α-helix bundle proteins with two radically different folds. Using a straightforward approach, we engineered the Sequences of one subdomain within maltose binding protein (MBP, α/β/α-sandwich) and another within outer surface protein A (OspA, β-sheet) to have high Sequence Identity (80 and 77%, respectively) with engineered variants of protein G (GA, 3-α-helix bundle). Circular dichroism and nuclear magnetic resonance spectra of all engineered variants demonstrate that they maintain their native conformations despite substantial Sequence modification. Furthermore, the MBP variant (80% identical to GA) remained active. Thermodynamic analysis of numerous GA and MBP variants suggests that the key to our approach involved stabilizing the modified MBP and OspA subdomains via external interactions with neighboring substructures, indicating that subdomain interactions can stabilize alternative folds over a broad range of Sequence variation. These findings suggest that it is possible to bridge one fold with many other topologies, which has implications for protein folding, evolution, and misfolding diseases.
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Subdomain interactions foster the design of two protein pairs with ∼80% Sequence Identity but different folds.
Biophysical journal, 2015Co-Authors: Lauren L Porter, Yihong Chen, John Orban, Philip N BryanAbstract:Metamorphic proteins, including proteins with high levels of Sequence Identity but different folds, are exceptions to the long-standing rule-of-thumb that proteins with as little as 30% Sequence Identity adopt the same fold. Which topologies can be bridged by these highly identical Sequences remains an open question. Here we bridge two 3-α-helix bundle proteins with two radically different folds. Using a straightforward approach, we engineered the Sequences of one subdomain within maltose binding protein (MBP, α/β/α-sandwich) and another within outer surface protein A (OspA, β-sheet) to have high Sequence Identity (80 and 77%, respectively) with engineered variants of protein G (GA, 3-α-helix bundle). Circular dichroism and nuclear magnetic resonance spectra of all engineered variants demonstrate that they maintain their native conformations despite substantial Sequence modification. Furthermore, the MBP variant (80% identical to GA) remained active. Thermodynamic analysis of numerous GA and MBP variants suggests that the key to our approach involved stabilizing the modified MBP and OspA subdomains via external interactions with neighboring substructures, indicating that subdomain interactions can stabilize alternative folds over a broad range of Sequence variation. These findings suggest that it is possible to bridge one fold with many other topologies, which has implications for protein folding, evolution, and misfolding diseases.
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de novo structure generation using chemical shifts for proteins with high Sequence Identity but different folds
Protein Science, 2010Co-Authors: Yang Shen, Philip N Bryan, John Orban, David Baker, Ad BaxAbstract:Proteins with high-Sequence Identity but very different folds present a special challenge to Sequence-based protein structure prediction methods. In particular, a 56-residue three-helical bundle protein (GA95) and an α/β-fold protein (GB95), which share 95% Sequence Identity, were targets in the CASP-8 structure prediction contest. With only 12 out of 300 submitted server-CASP8 models for GA95 exhibiting the correct fold, this protein proved particularly challenging despite its small size. Here, we demonstrate that the information contained in NMR chemical shifts can readily be exploited by the CS-Rosetta structure prediction program and yields adequate convergence, even when input chemical shifts are limited to just amide 1HN and 15N or 1HN and 1Hα values.
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nmr structures of two designed proteins with high Sequence Identity but different fold and function
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Yihong Chen, Patrick Alexander, Philip N Bryan, John OrbanAbstract:How protein Sequence codes for 3D structure remains a fundamental question in biology. One approach to understanding the folding code is to design a pair of proteins with maximal Sequence Identity but retaining different folds. Therefore, the nonidentities must be responsible for determining which fold topology prevails and constitute a fold-specific folding code. We recently designed two proteins, GA88 and GB88, with 88% Sequence Identity but different folds and functions [Alexander et al. (2007) Proc Natl Acad Sci USA 104:11963–11968]. Here, we describe the detailed 3D structures of these proteins determined in solution by NMR spectroscopy. Despite a large number of mutations taking the Sequence Identity level from 16 to 88%, GA88 and GB88 maintain their distinct wild-type 3-α and α/β folds, respectively. To our knowledge, the 3D-structure determination of two monomeric proteins with such high Sequence Identity but different fold topology is unprecedented. The geometries of the seven nonidentical residues (of 56 total) provide insights into the structural basis for switching between 3-α and α/β conformations. Further mutation of a subset of these nonidentities, guided by the GA88 and GB88 structures, leads to proteins with even higher levels of Sequence Identity (95%) and different folds. Thus, conformational switching to an alternative monomeric fold of comparable stability can be effected with just a handful of mutations in a small protein. This result has implications for understanding not only the folding code but also the evolution of new folds.
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the design and characterization of two proteins with 88 Sequence Identity but different structure and function
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Patrick Alexander, Yihong Chen, John Orban, Philip N BryanAbstract:To identify a simplified code for conformational switching, we have redesigned two natural proteins to have 88% Sequence Identity but different tertiary structures: a 3-α helix fold and an α/β fold. We describe the design of these homologous heteromorphic proteins, their structural properties as determined by NMR, their conformational stabilities, and their affinities for their respective ligands: IgG and serum albumin. Each of these proteins is completely folded at 25°C, is monomeric, and retains the native binding activity. The complete binding epitope for both ligands is encoded within each of the proteins. The IgG-binding epitope is functional only in the α/β fold, and the albumin-binding epitope is functional only in the 3-α fold. These results demonstrate that two monomeric folds and two different functions can be encoded with only 12% of the amino acids in a protein (7 of 56). The fact that 49 aa in these proteins are compatible with both folds shows that the essential information determining a fold can be highly concentrated in a few amino acids and that a very limited subset of interactions in the protein can tip the balance from one monomer fold to another. This delicate balance helps explain why protein structure prediction is so challenging. Furthermore, because a few mutations can result in both new conformation and new function, the evolution of new folds driven by natural selection for alternative functions may be much more probable than previously recognized.
Yihong Chen - One of the best experts on this subject based on the ideXlab platform.
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Subdomain interactions foster the design of two protein pairs with ∼80% Sequence Identity but different folds.
Biophysical journal, 2015Co-Authors: Lauren L Porter, Yihong Chen, John Orban, Philip N BryanAbstract:Metamorphic proteins, including proteins with high levels of Sequence Identity but different folds, are exceptions to the long-standing rule-of-thumb that proteins with as little as 30% Sequence Identity adopt the same fold. Which topologies can be bridged by these highly identical Sequences remains an open question. Here we bridge two 3-α-helix bundle proteins with two radically different folds. Using a straightforward approach, we engineered the Sequences of one subdomain within maltose binding protein (MBP, α/β/α-sandwich) and another within outer surface protein A (OspA, β-sheet) to have high Sequence Identity (80 and 77%, respectively) with engineered variants of protein G (GA, 3-α-helix bundle). Circular dichroism and nuclear magnetic resonance spectra of all engineered variants demonstrate that they maintain their native conformations despite substantial Sequence modification. Furthermore, the MBP variant (80% identical to GA) remained active. Thermodynamic analysis of numerous GA and MBP variants suggests that the key to our approach involved stabilizing the modified MBP and OspA subdomains via external interactions with neighboring substructures, indicating that subdomain interactions can stabilize alternative folds over a broad range of Sequence variation. These findings suggest that it is possible to bridge one fold with many other topologies, which has implications for protein folding, evolution, and misfolding diseases.
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subdomain interactions foster the design of two protein pairs with 80 Sequence Identity but different folds
Biophysical Journal, 2015Co-Authors: Lauren L Porter, Yihong Chen, John Orban, Philip N BryanAbstract:Metamorphic proteins, including proteins with high levels of Sequence Identity but different folds, are exceptions to the long-standing rule-of-thumb that proteins with as little as 30% Sequence Identity adopt the same fold. Which topologies can be bridged by these highly identical Sequences remains an open question. Here we bridge two 3-α-helix bundle proteins with two radically different folds. Using a straightforward approach, we engineered the Sequences of one subdomain within maltose binding protein (MBP, α/β/α-sandwich) and another within outer surface protein A (OspA, β-sheet) to have high Sequence Identity (80 and 77%, respectively) with engineered variants of protein G (GA, 3-α-helix bundle). Circular dichroism and nuclear magnetic resonance spectra of all engineered variants demonstrate that they maintain their native conformations despite substantial Sequence modification. Furthermore, the MBP variant (80% identical to GA) remained active. Thermodynamic analysis of numerous GA and MBP variants suggests that the key to our approach involved stabilizing the modified MBP and OspA subdomains via external interactions with neighboring substructures, indicating that subdomain interactions can stabilize alternative folds over a broad range of Sequence variation. These findings suggest that it is possible to bridge one fold with many other topologies, which has implications for protein folding, evolution, and misfolding diseases.
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nmr structures of two designed proteins with high Sequence Identity but different fold and function
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Yihong Chen, Patrick Alexander, Philip N Bryan, John OrbanAbstract:How protein Sequence codes for 3D structure remains a fundamental question in biology. One approach to understanding the folding code is to design a pair of proteins with maximal Sequence Identity but retaining different folds. Therefore, the nonidentities must be responsible for determining which fold topology prevails and constitute a fold-specific folding code. We recently designed two proteins, GA88 and GB88, with 88% Sequence Identity but different folds and functions [Alexander et al. (2007) Proc Natl Acad Sci USA 104:11963–11968]. Here, we describe the detailed 3D structures of these proteins determined in solution by NMR spectroscopy. Despite a large number of mutations taking the Sequence Identity level from 16 to 88%, GA88 and GB88 maintain their distinct wild-type 3-α and α/β folds, respectively. To our knowledge, the 3D-structure determination of two monomeric proteins with such high Sequence Identity but different fold topology is unprecedented. The geometries of the seven nonidentical residues (of 56 total) provide insights into the structural basis for switching between 3-α and α/β conformations. Further mutation of a subset of these nonidentities, guided by the GA88 and GB88 structures, leads to proteins with even higher levels of Sequence Identity (95%) and different folds. Thus, conformational switching to an alternative monomeric fold of comparable stability can be effected with just a handful of mutations in a small protein. This result has implications for understanding not only the folding code but also the evolution of new folds.
-
the design and characterization of two proteins with 88 Sequence Identity but different structure and function
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Patrick Alexander, Yihong Chen, John Orban, Philip N BryanAbstract:To identify a simplified code for conformational switching, we have redesigned two natural proteins to have 88% Sequence Identity but different tertiary structures: a 3-α helix fold and an α/β fold. We describe the design of these homologous heteromorphic proteins, their structural properties as determined by NMR, their conformational stabilities, and their affinities for their respective ligands: IgG and serum albumin. Each of these proteins is completely folded at 25°C, is monomeric, and retains the native binding activity. The complete binding epitope for both ligands is encoded within each of the proteins. The IgG-binding epitope is functional only in the α/β fold, and the albumin-binding epitope is functional only in the 3-α fold. These results demonstrate that two monomeric folds and two different functions can be encoded with only 12% of the amino acids in a protein (7 of 56). The fact that 49 aa in these proteins are compatible with both folds shows that the essential information determining a fold can be highly concentrated in a few amino acids and that a very limited subset of interactions in the protein can tip the balance from one monomer fold to another. This delicate balance helps explain why protein structure prediction is so challenging. Furthermore, because a few mutations can result in both new conformation and new function, the evolution of new folds driven by natural selection for alternative functions may be much more probable than previously recognized.
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The design and characterization of two proteins with 88% Sequence Identity but different structure and function
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Patrick Alexander, Yihong Chen, John Orban, Philip N BryanAbstract:To identify a simplified code for conformational switching, we have redesigned two natural proteins to have 88% Sequence Identity but different tertiary structures: a 3-α helix fold and an α/β fold. We describe the design of these homologous heteromorphic proteins, their structural properties as determined by NMR, their conformational stabilities, and their affinities for their respective ligands: IgG and serum albumin. Each of these proteins is completely folded at 25°C, is monomeric, and retains the native binding activity. The complete binding epitope for both ligands is encoded within each of the proteins. The IgG-binding epitope is functional only in the α/β fold, and the albumin-binding epitope is functional only in the 3-α fold. These results demonstrate that two monomeric folds and two different functions can be encoded with only 12% of the amino acids in a protein (7 of 56). The fact that 49 aa in these proteins are compatible with both folds shows that the essential information determining a fold can be highly concentrated in a few amino acids and that a very limited subset of interactions in the protein can tip the balance from one monomer fold to another. This delicate balance helps explain why protein structure prediction is so challenging. Furthermore, because a few mutations can result in both new conformation and new function, the evolution of new folds driven by natural selection for alternative functions may be much more probable than previously recognized.
B C Trauth - One of the best experts on this subject based on the ideXlab platform.
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purification and molecular cloning of the apo 1 cell surface antigen a member of the tumor necrosis factor nerve growth factor receptor superfamily Sequence Identity with the fas antigen
Journal of Biological Chemistry, 1992Co-Authors: A Oehm, Iris Behrmann, Werner Falk, Michael Pawlita, Gerhard Maier, C Klas, Min Liweber, S Richards, Jens Dhein, B C TrauthAbstract:Abstract The APO-1 antigen as defined by the mouse monoclonal antibody anti-APO-1 was previously found to be expressed on the cell surface of activated human T and B lymphocytes and a variety of malignant human lymphoid cell lines. Cross-linking of the APO-1 antigen by anti-APO-1 induced programmed cell death, apoptosis, of APO-1 positive cells. To characterize the APO-1 cell surface molecule and to better understand its role in induction of apoptosis, the APO-1 protein was purified to homogeneity from membranes of SKW6.4 B lymphoblastoid cells by solubilization with sodium deoxycholate, affinity chromatography with anti-APO-1 antibody, and reversed phase high performance liquid chromatography. Each purification step was followed by an APO-1-specific solid phase enzyme-linked immunosorbent assay using the monoclonal antibody anti-APO-1. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the APO-1 antigen was found to be a membrane glycoprotein of 48-kDa. Endoproteinase-cleaved peptides of the APO-1 protein were subjected to amino acid sequencing, and corresponding oligonucleotides were used to identify a full-length APO-1 cDNA clone from an SKW6.4 cDNA library. The deduced amino acid Sequence of APO-1 showed Sequence Identity with the Fas antigen, a cysteine-rich transmembrane protein of 335 amino acids with significant similarity to the members of the tumor necrosis factor/nerve growth factor receptor superfamily. The APO-1 antigen was expressed upon transfection of APO-1 cDNA into BL60-P7 Burkitt's lymphoma cells and conferred sensitivity towards anti-APO-1-induced apoptosis to the transfectants.
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Purification and molecular cloning of the APO-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor receptor superfamily. Sequence Identity with the Fas antigen.
The Journal of biological chemistry, 1992Co-Authors: A Oehm, Iris Behrmann, Werner Falk, Michael Pawlita, Gerhard Maier, C Klas, S Richards, Jens Dhein, Min Li-weber, B C TrauthAbstract:The APO-1 antigen as defined by the mouse monoclonal antibody anti-APO-1 was previously found to be expressed on the cell surface of activated human T and B lymphocytes and a variety of malignant human lymphoid cell lines. Cross-linking of the APO-1 antigen by anti-APO-1 induced programmed cell death, apoptosis, of APO-1 positive cells. To characterize the APO-1 cell surface molecule and to better understand its role in induction of apoptosis, the APO-1 protein was purified to homogeneity from membranes of SKW6.4 B lymphoblastoid cells by solubilization with sodium deoxycholate, affinity chromatography with anti-APO-1 antibody, and reversed phase high performance liquid chromatography. Each purification step was followed by an APO-1-specific solid phase enzyme-linked immunosorbent assay using the monoclonal antibody anti-APO-1. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the APO-1 antigen was found to be a membrane glycoprotein of 48-kDa. Endoproteinase-cleaved peptides of the APO-1 protein were subjected to amino acid sequencing, and corresponding oligonucleotides were used to identify a full-length APO-1 cDNA clone from an SKW6.4 cDNA library. The deduced amino acid Sequence of APO-1 showed Sequence Identity with the Fas antigen, a cysteine-rich transmembrane protein of 335 amino acids with significant similarity to the members of the tumor necrosis factor/nerve growth factor receptor superfamily. The APO-1 antigen was expressed upon transfection of APO-1 cDNA into BL60-P7 Burkitt's lymphoma cells and conferred sensitivity towards anti-APO-1-induced apoptosis to the transfectants.
Lauren L Porter - One of the best experts on this subject based on the ideXlab platform.
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subdomain interactions foster the design of two protein pairs with 80 Sequence Identity but different folds
Biophysical Journal, 2015Co-Authors: Lauren L Porter, Yihong Chen, John Orban, Philip N BryanAbstract:Metamorphic proteins, including proteins with high levels of Sequence Identity but different folds, are exceptions to the long-standing rule-of-thumb that proteins with as little as 30% Sequence Identity adopt the same fold. Which topologies can be bridged by these highly identical Sequences remains an open question. Here we bridge two 3-α-helix bundle proteins with two radically different folds. Using a straightforward approach, we engineered the Sequences of one subdomain within maltose binding protein (MBP, α/β/α-sandwich) and another within outer surface protein A (OspA, β-sheet) to have high Sequence Identity (80 and 77%, respectively) with engineered variants of protein G (GA, 3-α-helix bundle). Circular dichroism and nuclear magnetic resonance spectra of all engineered variants demonstrate that they maintain their native conformations despite substantial Sequence modification. Furthermore, the MBP variant (80% identical to GA) remained active. Thermodynamic analysis of numerous GA and MBP variants suggests that the key to our approach involved stabilizing the modified MBP and OspA subdomains via external interactions with neighboring substructures, indicating that subdomain interactions can stabilize alternative folds over a broad range of Sequence variation. These findings suggest that it is possible to bridge one fold with many other topologies, which has implications for protein folding, evolution, and misfolding diseases.
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Subdomain interactions foster the design of two protein pairs with ∼80% Sequence Identity but different folds.
Biophysical journal, 2015Co-Authors: Lauren L Porter, Yihong Chen, John Orban, Philip N BryanAbstract:Metamorphic proteins, including proteins with high levels of Sequence Identity but different folds, are exceptions to the long-standing rule-of-thumb that proteins with as little as 30% Sequence Identity adopt the same fold. Which topologies can be bridged by these highly identical Sequences remains an open question. Here we bridge two 3-α-helix bundle proteins with two radically different folds. Using a straightforward approach, we engineered the Sequences of one subdomain within maltose binding protein (MBP, α/β/α-sandwich) and another within outer surface protein A (OspA, β-sheet) to have high Sequence Identity (80 and 77%, respectively) with engineered variants of protein G (GA, 3-α-helix bundle). Circular dichroism and nuclear magnetic resonance spectra of all engineered variants demonstrate that they maintain their native conformations despite substantial Sequence modification. Furthermore, the MBP variant (80% identical to GA) remained active. Thermodynamic analysis of numerous GA and MBP variants suggests that the key to our approach involved stabilizing the modified MBP and OspA subdomains via external interactions with neighboring substructures, indicating that subdomain interactions can stabilize alternative folds over a broad range of Sequence variation. These findings suggest that it is possible to bridge one fold with many other topologies, which has implications for protein folding, evolution, and misfolding diseases.
