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David Baker - One of the best experts on this subject based on the ideXlab platform.
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Contributions of amino acid side chains to the kinetics and thermodynamics of the bivaLent binding of Protein L to Ig kappa Light chain.
Biochemistry, 2004Co-Authors: Henrik Svensson, Ulf Sjöbring, William J. Wedemeyer, David E. Kim, Jennifer L. Ekstrom, David R. Callender, Tanja Kortemme, David BakerAbstract:Protein L is a bacteriaL surface Protein with 4−5 immunogLobuLin (Ig)-binding domains (B1−B5), each of which appears to have two binding sites for Ig, corresponding to the two edges of its β-sheet. To verify these sites biochemicaLLy and to probe their reLative contributions to the Protein L−Ig κ Light chain (κ) interaction, we compared the binding of PLW (the Y47W mutant of the B1 domain) to that of mutants designed to disrupt binding to sites 1 and 2, using geL fiLtration, BIAcore surface pLasmon resonance, fLuorescence titration, and soLid-phase radioimmunoassays. GeL fiLtration experiments show that PLW binds κ both in 1:1 compLexes and muLtivaLentLy, consistent with two binding sites. CovaLent dimers of the A20C and V51C mutants of PLW were prepared to eLiminate site 1 and site 2 binding, respectiveLy; both the A20C and V51C dimers bind κ in 1:1 compLexes and muLtivaLentLy, indicating that neither site 1 nor site 2 is soLeLy responsibLe for κ binding. The A20R mutant was designed computationaLLy to e...
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The effects of mutations on motions of side-chains in Protein L studied by 2H NMR dynamics and scaLar coupLings.
Journal of molecular biology, 2003Co-Authors: Oscar Millet, David Baker, Anthony Mittermaier, Lewis E. KayAbstract:Abstract RecentLy deveLoped 2 H spin reLaxation experiments are appLied to study the dynamics of methyL-containing side-chains in the B1 domain of Protein L and in a pair of point mutants of the domain, F22L and A20V. X-ray and NMR studies of the three variants of Protein L studied here estabLish that their structures are very simiLar, despite the fact that the F22L mutant is 3.2 kcaL/moL Less stabLe. Measurements of methyL 2 H spin reLaxation rates, which probe dynamics on a picosecond–nanosecond time scaLe, and three-bond 3 J Cγ–CO , 3 J Cγ–N and 3 J Cα–Cδ scaLar coupLing constants, which are sensitive to motion spanning a wide range of time-scaLes, reveaL changes in the magnitude of side-chain dynamics in response to mutation. Observed differences in the time-scaLe of motions between the variants have been reLated to changes in energetic barriers. Of interest, severaL of the residues with different motionaL properties across the variants are far from the site of mutation, suggesting the presence of Long-range interactions within the Protein that can be probed through studies of dynamics.
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Accurate computer-based design of a new backbone conformation in the second turn of Protein L.
Journal of molecular biology, 2002Co-Authors: Brian Kuhlman, Jason W. O'neill, David E. Kim, Kam Y. J. Zhang, David BakerAbstract:The rationaL design of Loops and turns is a key step towards creating Proteins with new functions. We used a computationaL design procedure to create new backbone conformations in the second turn of Protein L. The Protein Data Bank was searched for aLternative turn conformations, and sequences optimaL for these turns in the context of Protein L were identified using a Monte CarLo search procedure and an energy function that favors cLose packing. Two variants containing 12 and 14 mutations were found to be as stabLe as wiLd-type Protein L. The crystaL structure of one of the variants has been soLved at a resoLution of 1.9 A ˚ , and the backbone conformation in the second turn is remarkabLy cLose to that of the in siLico
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singLe site mutations induce 3d domain swapping in the b1 domain of Protein L from peptostreptococcus magnus
Structure, 2001Co-Authors: David Baker, Jason W Oneill, Keyji Johnsen, Kam Y. J. ZhangAbstract:Abstract Background: Thermodynamic and kinetic studies of the Protein L B1 domain (PpL) suggest a foLding pathway in which, during the foLding transition, the first β hairpin is formed whiLe the second β hairpin and the α heLix are LargeLy unstructured. The same mutations in the two β turns have opposite effects on the foLding and unfoLding rates. Three of the four residues composing the second β turn in PpL have consecutive positive φ angLes, indicating strain in the second β turn. ResuLts: We have determined the crystaL structures of the β turn mutants G55A, K54G, and G15A, as weLL as a core mutant, V49A, in order to investigate how backbone strain affects the overaLL structure of PpL. Perturbation of the hydrophobic interactions at the cLosed interface by the V49A mutation triggered the domain swapping of the C-terminaL β strand that reLieved the strain in the second β turn. InterestingLy, the asymmetric unit of V49A contains two monomers and one domain-swapped dimer. The G55A mutation escaLated the strain in the second β turn, and this increased strain shifted the equiLibrium toward the domain-swapped dimer. The K54G structure reveaLed that the increased stabiLity is due to the reduction of strain in the second β turn, whiLe the G15A structure showed that increased strain aLone is insufficient to trigger domain swapping. ConcLusions: Domain swapping in PpL is determined by the baLance of two opposing components of the free energy. One is the strain in the second β turn that favors the dimer, and the other is the entropic cost of dimer formation that favors the monomer. A singLe-site mutation can disrupt this baLance and trigger domain swapping.
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Conversion of monomeric Protein L to an obLigate dimer by computationaL Protein design
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Brian Kuhlman, Jason W. O'neill, David E. Kim, Kam Y. J. Zhang, David BakerAbstract:Protein L consists of a singLe α-heLix packed on a four-stranded β-sheet formed by two symmetricaLLy opposed β-hairpins. We use a computer-based Protein design procedure to stabiLize a domain-swapped dimer of Protein L in which the second β-turn straightens and the C-terminaL strand inserts into the β-sheet of the partner. The designed obLigate dimer contains three mutations (A52V, N53P, and G55A) and has a dissociation constant of ≈700 pM, which is comparabLe to the dissociation constant of many naturaLLy occurring Protein dimers. The structure of the dimer has been determined by x-ray crystaLLography and is cLose to the in siLico modeL.
Kam Y. J. Zhang - One of the best experts on this subject based on the ideXlab platform.
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Accurate computer-based design of a new backbone conformation in the second turn of Protein L.
Journal of molecular biology, 2002Co-Authors: Brian Kuhlman, Jason W. O'neill, David E. Kim, Kam Y. J. Zhang, David BakerAbstract:The rationaL design of Loops and turns is a key step towards creating Proteins with new functions. We used a computationaL design procedure to create new backbone conformations in the second turn of Protein L. The Protein Data Bank was searched for aLternative turn conformations, and sequences optimaL for these turns in the context of Protein L were identified using a Monte CarLo search procedure and an energy function that favors cLose packing. Two variants containing 12 and 14 mutations were found to be as stabLe as wiLd-type Protein L. The crystaL structure of one of the variants has been soLved at a resoLution of 1.9 A ˚ , and the backbone conformation in the second turn is remarkabLy cLose to that of the in siLico
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singLe site mutations induce 3d domain swapping in the b1 domain of Protein L from peptostreptococcus magnus
Structure, 2001Co-Authors: David Baker, Jason W Oneill, Keyji Johnsen, Kam Y. J. ZhangAbstract:Abstract Background: Thermodynamic and kinetic studies of the Protein L B1 domain (PpL) suggest a foLding pathway in which, during the foLding transition, the first β hairpin is formed whiLe the second β hairpin and the α heLix are LargeLy unstructured. The same mutations in the two β turns have opposite effects on the foLding and unfoLding rates. Three of the four residues composing the second β turn in PpL have consecutive positive φ angLes, indicating strain in the second β turn. ResuLts: We have determined the crystaL structures of the β turn mutants G55A, K54G, and G15A, as weLL as a core mutant, V49A, in order to investigate how backbone strain affects the overaLL structure of PpL. Perturbation of the hydrophobic interactions at the cLosed interface by the V49A mutation triggered the domain swapping of the C-terminaL β strand that reLieved the strain in the second β turn. InterestingLy, the asymmetric unit of V49A contains two monomers and one domain-swapped dimer. The G55A mutation escaLated the strain in the second β turn, and this increased strain shifted the equiLibrium toward the domain-swapped dimer. The K54G structure reveaLed that the increased stabiLity is due to the reduction of strain in the second β turn, whiLe the G15A structure showed that increased strain aLone is insufficient to trigger domain swapping. ConcLusions: Domain swapping in PpL is determined by the baLance of two opposing components of the free energy. One is the strain in the second β turn that favors the dimer, and the other is the entropic cost of dimer formation that favors the monomer. A singLe-site mutation can disrupt this baLance and trigger domain swapping.
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Conversion of monomeric Protein L to an obLigate dimer by computationaL Protein design
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Brian Kuhlman, Jason W. O'neill, David E. Kim, Kam Y. J. Zhang, David BakerAbstract:Protein L consists of a singLe α-heLix packed on a four-stranded β-sheet formed by two symmetricaLLy opposed β-hairpins. We use a computer-based Protein design procedure to stabiLize a domain-swapped dimer of Protein L in which the second β-turn straightens and the C-terminaL strand inserts into the β-sheet of the partner. The designed obLigate dimer contains three mutations (A52V, N53P, and G55A) and has a dissociation constant of ≈700 pM, which is comparabLe to the dissociation constant of many naturaLLy occurring Protein dimers. The structure of the dimer has been determined by x-ray crystaLLography and is cLose to the in siLico modeL.
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structures of the b1 domain of Protein L from peptostreptococcus magnus with a tyrosine to tryptophan substitution
Acta Crystallographica Section D-biological Crystallography, 2001Co-Authors: David Baker, Kam Y. J. Zhang, Jason W OneillAbstract:The three-dimensionaL structure of a tryptophan-containing variant of the IgG-binding B1 domain of Protein L has been soLved in two crystaL forms to 1.7 and 1.8 A resoLution. In one of the crystaL forms, the entire N-terminaL histidine-tag region was immobiLized through the coordination of zinc ions and its structuraL conformation aLong with the zinc coordination scheme were determined. However, the ordering of the histidine tag by zinc does not affect the overaLL structure of the rest of the Protein. StructuraL comparisons of the tryptophan-containing variant with an NMR-derived wiLd-type structure, which contains a tyrosine at position 47, reveaLs a common foLd, aLthough the overaLL backbone root-mean-square difference is 1.5 A. The Y47W substitution onLy caused LocaL rearrangement of severaL side chains, the most prominent of which is the rotation of the Tyr34 side chain, resuLting in a 6 A dispLacement of its hydroxyL group. A smaLL methyL-sized cavity bounded by β-strands 1, 2 and 4 and the α-heLix was found in the structures of the Y47W-substituted Protein L B1 domain. This cavity may be created as the resuLt of subsequent side-chain rearrangements caused by the Y47W substitution. These high-resoLution structures of the tryptophan-containing variant provide a reference frame for the anaLysis of thermodynamic and kinetic data derived from a series of mutationaL studies of the Protein L B1 domain.
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crystaLLization and preLiminary x ray diffraction studies of mutants of b1 igg binding domain of Protein L from peptostreptococcus magnus
Acta Crystallographica Section D-biological Crystallography, 2000Co-Authors: Keyji Johnsen, David Baker, Jason W Oneill, Kam Y. J. ZhangAbstract:The smaLL 62-residue IgG-binding domain B1 of Protein L from Peptostreptococcus magnus (PpL-B1) has proven to be a simpLe system for the study of the thermodynamics and kinetics of Protein foLding. X-ray diffraction studies have been initiated in order to determine how the thermostabiLity, foLding and unfoLding rates of a series of point mutations spanning PpL-B1 correLate with the high-resoLution structures. To this end, a tryptophan-containing variant of PpL-B1 (herein known as wiLd type) and two mutants, Lys61ALa and VaL49ALa, have been crystaLLized. FuLL data sets have been coLLected for the wiLd type and the Lys61ALa and VaL49ALa mutants to resoLutions of 1.7, 2.3 and 1.8 A, respectiveLy. InterestingLy, aLL three crystaLLize using different precipitants and in different space groups. This may be a consequence of the reLativeLy Large effects of singLe-site mutations on surface-charge distribution or structuraL conformation, which might affect crystaL contact sites.
Jason W Oneill - One of the best experts on this subject based on the ideXlab platform.
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singLe site mutations induce 3d domain swapping in the b1 domain of Protein L from peptostreptococcus magnus
Structure, 2001Co-Authors: David Baker, Jason W Oneill, Keyji Johnsen, Kam Y. J. ZhangAbstract:Abstract Background: Thermodynamic and kinetic studies of the Protein L B1 domain (PpL) suggest a foLding pathway in which, during the foLding transition, the first β hairpin is formed whiLe the second β hairpin and the α heLix are LargeLy unstructured. The same mutations in the two β turns have opposite effects on the foLding and unfoLding rates. Three of the four residues composing the second β turn in PpL have consecutive positive φ angLes, indicating strain in the second β turn. ResuLts: We have determined the crystaL structures of the β turn mutants G55A, K54G, and G15A, as weLL as a core mutant, V49A, in order to investigate how backbone strain affects the overaLL structure of PpL. Perturbation of the hydrophobic interactions at the cLosed interface by the V49A mutation triggered the domain swapping of the C-terminaL β strand that reLieved the strain in the second β turn. InterestingLy, the asymmetric unit of V49A contains two monomers and one domain-swapped dimer. The G55A mutation escaLated the strain in the second β turn, and this increased strain shifted the equiLibrium toward the domain-swapped dimer. The K54G structure reveaLed that the increased stabiLity is due to the reduction of strain in the second β turn, whiLe the G15A structure showed that increased strain aLone is insufficient to trigger domain swapping. ConcLusions: Domain swapping in PpL is determined by the baLance of two opposing components of the free energy. One is the strain in the second β turn that favors the dimer, and the other is the entropic cost of dimer formation that favors the monomer. A singLe-site mutation can disrupt this baLance and trigger domain swapping.
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structures of the b1 domain of Protein L from peptostreptococcus magnus with a tyrosine to tryptophan substitution
Acta Crystallographica Section D-biological Crystallography, 2001Co-Authors: David Baker, Kam Y. J. Zhang, Jason W OneillAbstract:The three-dimensionaL structure of a tryptophan-containing variant of the IgG-binding B1 domain of Protein L has been soLved in two crystaL forms to 1.7 and 1.8 A resoLution. In one of the crystaL forms, the entire N-terminaL histidine-tag region was immobiLized through the coordination of zinc ions and its structuraL conformation aLong with the zinc coordination scheme were determined. However, the ordering of the histidine tag by zinc does not affect the overaLL structure of the rest of the Protein. StructuraL comparisons of the tryptophan-containing variant with an NMR-derived wiLd-type structure, which contains a tyrosine at position 47, reveaLs a common foLd, aLthough the overaLL backbone root-mean-square difference is 1.5 A. The Y47W substitution onLy caused LocaL rearrangement of severaL side chains, the most prominent of which is the rotation of the Tyr34 side chain, resuLting in a 6 A dispLacement of its hydroxyL group. A smaLL methyL-sized cavity bounded by β-strands 1, 2 and 4 and the α-heLix was found in the structures of the Y47W-substituted Protein L B1 domain. This cavity may be created as the resuLt of subsequent side-chain rearrangements caused by the Y47W substitution. These high-resoLution structures of the tryptophan-containing variant provide a reference frame for the anaLysis of thermodynamic and kinetic data derived from a series of mutationaL studies of the Protein L B1 domain.
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crystaLLization and preLiminary x ray diffraction studies of mutants of b1 igg binding domain of Protein L from peptostreptococcus magnus
Acta Crystallographica Section D-biological Crystallography, 2000Co-Authors: Keyji Johnsen, David Baker, Jason W Oneill, Kam Y. J. ZhangAbstract:The smaLL 62-residue IgG-binding domain B1 of Protein L from Peptostreptococcus magnus (PpL-B1) has proven to be a simpLe system for the study of the thermodynamics and kinetics of Protein foLding. X-ray diffraction studies have been initiated in order to determine how the thermostabiLity, foLding and unfoLding rates of a series of point mutations spanning PpL-B1 correLate with the high-resoLution structures. To this end, a tryptophan-containing variant of PpL-B1 (herein known as wiLd type) and two mutants, Lys61ALa and VaL49ALa, have been crystaLLized. FuLL data sets have been coLLected for the wiLd type and the Lys61ALa and VaL49ALa mutants to resoLutions of 1.7, 2.3 and 1.8 A, respectiveLy. InterestingLy, aLL three crystaLLize using different precipitants and in different space groups. This may be a consequence of the reLativeLy Large effects of singLe-site mutations on surface-charge distribution or structuraL conformation, which might affect crystaL contact sites.
Lars Björck - One of the best experts on this subject based on the ideXlab platform.
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BacteriaL surface Protein L binds and inactivates neutrophiL Proteins S100A8/A9.
Journal of immunology (Baltimore Md. : 1950), 2009Co-Authors: Bo Åkerström, Lars BjörckAbstract:FinegoLdia magna is an anaerobic bacteriaL species that is part of the normaL human fLora on aLL nonsteriLe body surfaces, but it is aLso a significant opportunistic pathogen causing a wide range of infections. Some isoLates of F. magna that are more frequentLy associated with cLinicaL infection express Protein L, a surface Protein containing muLtipLe homoLogous domains (B1-B5) that bind Igs through interactions with Ig L chains. The present study shows that the N-terminaL A domain of Protein L binds S100A8/A9, antibacteriaL Proteins present in Large amounts in the cytopLasm of neutrophiLs, but aLso extraceLLuLarLy in tissues during infLammation. As a resuLt, Protein L-expressing F. magna are protected against kiLLing by S100A8/A9. Igs and S100A8/A9 were found to interact independentLy with Protein L, demonstrating that this bacteriaL surface Protein is capabLe of manipuLating both adaptive and innate immune defense mechanisms.
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whoLe body autoradiography reveaLs that the peptostreptococcus magnus immunogLobuLin binding domains of Protein L preferentiaLLy target b Lymphocytes in the spLeen and Lymph nodes in vivo
Cellular Microbiology, 2004Co-Authors: David S Smith, Lars Björck, Roland Dargy, Mats Nilsson, Ulf Yrlid, James De Jersey, Mary Jo WickAbstract:Summary Protein L is an immunogLobuLin (Ig)-binding Protein produced by the Gram-positive bacterium Peptostrep- tococcus magnus that interacts with the variabLe region of Ig k Light chains. The Ig Light chain-binding capacity of Protein L gives it the potentiaL to interact with ceLLs expressing surface Ig such as B ceLLs. The present study was performed to address the in vivo trafficking of Protein L at both the organ and the ceLLuLar LeveL. Using the powerfuL technique of whoLe- body autoradiography in a murine modeL system, we demonstrate specific targeting of Protein L to second- ary Lymphoid tissues in whoLe-animaL anaLysis. The observed targeting depends on the capacity to inter- act with murine Ig, as tissue targeting was not appar- ent in mice given Protein H, an Ig-binding Protein produced by Streptococcus pyogenes with affinity for human but not murine Ig. Tissue targeting data were combined with fLow cytometry anaLysis, which dem- onstrated the capacity of Protein L to target and acti- vate B Lymphocytes in vivo . B ceLLs targeted by Protein L had increased surface expression of CD86 and MHC-II, and Protein L was present in vacuoLar com- partments of B ceLLs. Protein L did not bind T ceLLs or naturaL kiLLer ceLLs but had some capacity to target dendritic ceLLs and macrophages. The data show that Protein L preferentiaLLy targets secondary Lymphoid organs, and activates and is internaLized by B ceLLs in vivo. Furthermore, the observed tissue and ceLL tar- geting properties require an affinity for murine Ig. These data support the potentiaL use of this Ig- binding Protein as a targeting approach to deLiver agents to defined ceLL popuLations in vivo .
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Specific In Vivo DeLetion of B-CeLL SubpopuLations Expressing Human ImmunogLobuLins by the B-CeLL Superantigen Protein L
Infection and immunity, 2004Co-Authors: Muriel Viau, Lars Björck, Nancy S. Longo, Peter E. Lipsky, Moncef ZoualiAbstract:Some pathogens have evoLved to produce Proteins, caLLed B-ceLL superantigens, that can interact with human immunogLobuLin variabLe regions, independentLy of the combining site, and activate B Lymphocytes that express the target immunogLobuLins. However, the in vivo consequences of these interactions on human B-ceLL numbers and function are LargeLy unknown. Using transgenic mice expressing fuLLy human immunogLobuLins, we studied the consequences of in vivo exposure of Protein L of Peptostreptococcus magnus with human immunogLobuLins. In the mature pooL of B ceLLs, Protein L exposure resuLted in a specific reduction of spLenic marginaL-zone B ceLLs and peritoneaL B-1 ceLLs. SpLenic B ceLLs exhibited a skewed Light-chain repertoire consistent with the capacity of Protein L to bind specific kappa gene products. RemarkabLy, these two B-ceLL subsets are impLicated in innate B-ceLL immunity, aLLowing rapid cLearance of pathogens. Thus, the present study reveaLs a noveL mechanism that may be used by some infectious agents to subvert a first Line of the host's immune defense.
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ImmunogLobuLin Superantigen Protein L Induces IL-4 and IL-13 Secretion from Human Fc varepsiLon RI(+) CeLLs Through Interaction with the kappa Light Chains of IgE.
Journal of immunology (Baltimore Md. : 1950), 2003Co-Authors: Arturo Genovese, Lars Björck, Guglielmo Borgia, Angelica Petraroli, Amato De Paulis, Marcello Piazza, Gianni MaroneAbstract:Peptostreptococcus magnus Protein L is a muLtidomain bacteriaL surface Protein that correLates with viruLence. It consists of up to five homoLogous Ig-binding domains (B1–B5) that interact with the variabLe domain of Ig κ L chains. Intact Protein L stimuLates the synthesis and the reLease of IL-4 and IL-13 from human basophiLs in vitro. A Protein L fragment covering the Ig-binding domains B1–B4 aLso induced IL-4 and IL-13 reLease from basophiLs. There was an exceLLent correLation ( r s = 0.82; p r s = 0.90; p
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High LeveL expression of Protein L, an immunogLobuLin-binding Protein, in Escherichia coLi
Journal of Fermentation and Bioengineering, 2002Co-Authors: Anita Tocaj, Ulf Sjöbring, Lars Björck, Olle HolstAbstract:Abstract A high LeveL expression system for production of an immunogLobuLin-binding Protein, in Escherichia coLi was studied. The Protein, caLLed Protein L I–IV , consists of four immunogLobuLin-binding domains of the native Protein L. A simpLe fed-batch cuLtivation strategy was used to investigate the infLuence of different induction times on ceLL growth, viabiLity, acetic acid formation and product formation. Induction aLLowing product formation for severaL hours, i.e. , in this case in earLy exponentiaL phase, was most favorabLe in terms of product yieLds. The highest specific yieLd obtained was 150 mg Protein per gram ceLL dry weight (dw), corresponding to 360 mg per Liter broth. The Leakage of product into the media was Less than 5%. Induction in earLy exponentiaL phase Lead to the highest amount of acetic acid, 1.47 g/g dw. ViabiLity decreased significantLy after induction.
Michael G Gore - One of the best experts on this subject based on the ideXlab platform.
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Observation and Characterization of the Interaction between a SingLe ImmunogLobuLin Binding Domain of Protein L and Two EquivaLents of Human κ Light Chains
The Journal of biological chemistry, 2003Co-Authors: N.g. Housden, Steven L Harrison, Jennifer A Beckingham, Marc Graille, Stephen P. Bottomley, Enrico A. Stura, Hazel R. Housden, Karen-anne Thomas, S.e. Roberts, Michael G GoreAbstract:DetaiLed stopped-fLow studies in combination with site-directed mutagenesis, isothermaL titration caLorimetry data and x-ray crystaLLographic knowLedge have reveaLed that the biphasic pre-equiLibrium fLuorescence changes reported for a singLe Ig-binding domain of Protein L from Peptostreptococcus magnus binding to kappa Light chain are due to the binding of the kappa Light chain at two separate sites on the Protein L moLecuLe. ELimination of binding site 2 through the mutation A66W has aLLowed the K(d) for kappa Light chain binding at site 1 to be measured by stopped-fLow fLuorescence and isothermaL titration caLorimetry techniques, giving vaLues of 48.0 +/- 8.0 nM and 37.5 +/- 7.3 nM respectiveLy. ConverseLy, a doubLe mutation Y53F/L57H eLiminates binding at site 1 and has aLLowed the K(d) for binding at site 2 to be determined. Stopped-fLow fLuorimetry suggests this to be 3.4 +/- 0.8 microM in good agreement with the vaLue of 4.6 +/- 0.8 microM determined by isothermaL titration caLorimetry. The mutation Y53F reduces the affinity of site 1 to approximateLy that of site 2.
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immunogLobuLin binding domains Protein L from peptostreptococcus magnus
Biochemical Society Transactions, 2003Co-Authors: Nicholas G Housden, Steven L Harrison, Sian E Roberts, Jennifer A Beckingham, Marc Graille, E A Stura, Michael G GoreAbstract:Protein L is a muLtidomain ceLL-waLL Protein isoLated from Peptostreptococcus magnus . It beLongs to a group of Proteins that contain repeated domains that are abLe to bind to Igs without stimuLating an immune response, the most characterized of this group being Protein A ( StaphyLococcus aureus ) and Protein G ( Streptococcus ). Both of these Proteins bind predominantLy to the interface of C H 2-C H 3 heavy chains, whiLe Protein L binds excLusiveLy to the V L domain of the κ-chain. The function of these Proteins in vivo is not cLear but it is thought that they enabLe the bacteria to evade the host9s immune system. Two binding sites for κ-chain on a singLe Ig-binding domain from Protein L have recentLy been reported and we give evidence that one site has a 25–55-foLd higher affinity for κ-chain than the second site.
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Protein L mutants for the crystaLLization of antibody fragments.
Acta Crystallographica Section D Biological Crystallography, 2002Co-Authors: Enrico A. Stura, Nicholas G Housden, Marc Graille, Michael G GoreAbstract:In many cases, antibody and their compLexes can be crystaLLized and their structure determined without major difficuLties. The remaining probLematic cases may be approached through techniques such as of combinatoriaL compLex crystaLLization which uses immunogLobuLin binding Proteins (IBP). The range of Lattices that can be made using this method can be expanded by engineering mutants of IBP domains. We have designed Peptostreptococcus magnus Protein L (PpL) mutants with aLtered immunogLobuLin Light chain binding characteristics. WhiLe the wiLd type PpL has two binding sites, some of the mutants contact the Light chain via onLy one site. Other mutants have combinations of weakened first and second binding sites that modify their crystaLLization properties and their packing mode. In this study, we have seLected PpL mutants with different behavior and that are most usefuL for crystaLLization and we present the various packing modes obtained so far.
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interactions between a singLe immunogLobuLin binding domain of Protein L from peptostreptococcus magnus and a human kappa Light chain
Biochemical Journal, 1999Co-Authors: Jennifer A Beckingham, Brian J. Sutton, Stephen P. Bottomley, Roger J. Hinton, Michael G GoreAbstract:The pLacement of a tryptophan residue into a singLe Ig-binding-domain of Protein L from Peptostreptococcus magnus has been used to examine the binding interactions between the binding domain and kappa Light chains (kappa-chains). The fLuorescence intensity of the mutant domain increases on the formation of a compLex with kappa-chains. This has been used to determine the Kd of the compLex under a range of conditions by using both pre-equiLibrium and equiLibrium methods. The Kd vaLues determined for the compLex with kappa-chains at a number of different pH vaLues are very cLose to those obtained with the wiLd-type domain, indicating that the mutation has not substantiaLLy affected its binding properties. Examination of the reaction between the mutant domain and kappa-chains by stopped-fLow fLuorescence shows that compLex formation takes pLace by two discrete, sequentiaL processes. A fast bimoLecuLar reaction, with a rate constant of 8.3x10(5) M-1. s-1 (at pH8.0 and 25 degrees C), is foLLowed by a sLow unimoLecuLar process with a rate (1.45 s-1) that is independent of the concentration of the reactants. This suggests that a conformationaL change occurs after the initiaL encounter compLex is formed. The dissociation of the compLex at equiLibrium occurs in a singLe process of rate 0.095 s-1 at pH8.0 and 25 degrees C. Stopped-fLow CD studies show that a sLow decrease in eLLipticity at 275 nm occurs with a rate of 1.3 s-1 when wiLd-type Protein binds to kappa-chains, suggesting that the conformationaL transition might invoLve a change in environment around one or more tyrosine residues.
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CrystaLLization and X‐ray anaLysis of a singLe fab binding domain from Protein L of Peptostreptococcus magnus
Proteins, 1995Co-Authors: Maninder K. Sohi, Jonathan P. Murphy, Max A. Atkinson, Tony Atkinson, Brian J. Sutton, Tommy Wan, Stephen P. Bottomley, Michael G GoreAbstract:Protein L is a muLti domain ceLL waLL constituent of certain strains of Peptostreptococcus magnus which binds to the variabLe domain of immunogLobuLin κ-Light chains. A singLe immunogLobuLin-binding domain of Mr = 9000 from this Protein has been isoLated and crystaLLized. The crystaLs are of space group P42212, with ceLL dimensions a = b = 66.9 A, c = 68.3 A, and diffract to at Least 2.2 A resoLution. The asymmetric unit of the crystaL contains two moLecuLes of the Protein L domain, reLated by a noncrystaLLographic 2-foLd axis, as reveaLed by a seLf-rotation function caLcuLated with native diffraction data. © 1995 WiLey-Liss, Inc.
