Ertan Eryilmaz - Academia.edu (original) (raw)
Papers by Ertan Eryilmaz
J Mol Biol, 2008
Cystoviruses are a class of enveloped double-stranded RNA viruses that use a multiprotein polymer... more Cystoviruses are a class of enveloped double-stranded RNA viruses that use a multiprotein polymerase complex (PX) to replicate and transcribe the viral genome. Although the structures of the polymerase and ATPase components of the cystoviral PX are known and their functional behavior is understood to a large extent, no atomic-resolution structural information is available for the major capsid protein P1 that defines the overall structure and symmetry of the viral capsid and the essential protein P7. Toward obtaining a complete structural and functional understanding of the cystoviral PX, we have obtained the structure of P7 from the cystovirus ϕ12 at a resolution of 1.8 Å. The N-terminal core region (1-129) of P7 forms a novel homodimeric α/β-fold having structural similarities with BRCT domains implicated in multiple protein-protein interactions in DNA repair proteins. Our results, combined with the known role of P7 in stabilizing the nucleation complex during capsid assembly, hint toward its participation in key protein-protein interactions within the cystoviral PX. Additionally, we have found through solution NMR studies that the C-terminal tail of P7 (130-169) that is essential for virus viability, although highly disordered, contains a nascent helix. We demonstrate for the first time, through NMR titrations, that P7 is capable of interacting with RNA. We find that both the N-terminal core and the dynamic C-terminal tail of P7 play a role in RNA recognition. This interaction leads to a significant reduction of the degree of disorder in the C-terminal tail. Given the requirement of P7 in maintaining genome packaging efficiency and transcriptional fidelity, our data suggest a central biological role for P7-RNA interactions.
Journal of Molecular Biology, 2009
Molecular Immunology, 2016
Anti-DNA antibodies are the serological hallmark of systemic lupus erythematosus, and participate... more Anti-DNA antibodies are the serological hallmark of systemic lupus erythematosus, and participate in the pathogenesis of lupus nephritis by cross-reacting with multiple renal antigens. Previously, using a panel of murine anti-DNA IgGs that share identical variable regions but that differ in the constant regions, we demonstrated that the cross-reaction and renal pathogenicity of anti-DNA antibodies are isotype dependent. In this study, we investigated the catalytic potential of this anti-DNA antibody panel, and determined its isotype dependency. The three isotype switch variants (IgG1, IgG2a, IgG2b) and the parent IgG3 PL9-11 anti-DNA antibodies were compared in their catalysis of 500 base pair linear double stranded DNA and a 12-mer peptide (ALWPPNLHAWVP), by gel analysis, MALDI-TOF mass spectrometry, and nuclear magnetic resonance spectroscopy. The binding affinity of anti-DNA antibodies to double stranded DNA and peptide antigens were assessed by ELISA and surface plasmon resonance. We found that the PL9-11 antibody isotypes vary significantly in their potential to catalyze the cleavage of both linear and double stranded DNA and the proteolysis of peptides. The degree of the cleavage and proteolysis increases with the incubation temperature and time. While different PL9-11 isotypes have the same initial attack sites within the ALWPPNLHAWVP peptide, there was no correlation between binding affinity to the peptide and proteolysis rates. In conclusion, the catalytic properties of anti-DNA antibodies are isotype dependent. This finding provides further evidence that antibodies that share the same variable region, but which have different constant regions, are functionally distinct. The catalytic effects modulated by antibody constant regions need to be considered in the design of therapeutic antibodies (abzymes) and peptides designed to block pathogenic autoantibodies.
Clinical and experimental immunology, Jan 20, 2015
Anti-DNA antibodies play a pivotal role in the pathogenesis of lupus nephritis by cross reacting ... more Anti-DNA antibodies play a pivotal role in the pathogenesis of lupus nephritis by cross reacting with renal antigens. Previously, we demonstrated that the binding affinity of anti-DNA antibodies to self-antigens is isotype dependent. Furthermore, significant variability in renal pathogenicity was seen among a panel of anti-DNA isotypes (derived from a single murine IgG3 monoclonal antibody, PL9-11) that share identical variable regions. In this study, we sought to select peptide mimics that effectively inhibit the binding of all murine and human anti-DNA IgG isotypes to glomerular antigens. The PL9-11 panel of IgG anti-DNA antibodies (IgG1, IgG2a, IgG2b, and IgG3) was used for screening a 12-mer phage display library. Binding affinity was determined by surface plasmon resonance. ELISA, flow cytometry, and glomerular binding assays were used for the assessment of peptide inhibition of antibody binding to nuclear and kidney antigens. We identified a 12 amino acid peptide (ALWPPNLHAWVP...
Clinical & Experimental Immunology, 2015
Anti-DNA antibodies play a pivotal role in the pathogenesis of lupus nephritis by cross reacting ... more Anti-DNA antibodies play a pivotal role in the pathogenesis of lupus nephritis by cross reacting with renal antigens. Previously, we demonstrated that the binding affinity of anti-DNA antibodies to self-antigens is isotype dependent. Furthermore, significant variability in renal pathogenicity was seen among a panel of anti-DNA isotypes (derived from a single murine IgG3 monoclonal antibody, PL9-11) that share identical variable regions. In this study, we sought to select peptide mimics that effectively inhibit the binding of all murine and human anti-DNA IgG isotypes to glomerular antigens. The PL9-11 panel of IgG anti-DNA antibodies (IgG1, IgG2a, IgG2b, and IgG3) was used for screening a 12-mer phage display library. Binding affinity was determined by surface plasmon resonance. ELISA, flow cytometry, and glomerular binding assays were used for the assessment of peptide inhibition of antibody binding to nuclear and kidney antigens. We identified a 12 amino acid peptide (ALWPPNLHAWVP, or "ALW") which binds to all PL9-11 IgG isotypes. Pre-incubation with the ALW peptide significantly reduced the binding of the PL9-11 anti-DNA antibodies to DNA, laminin, mesangial cells, and isolated glomeruli. Furthermore, we confirmed the specificity of the amino acid sequence in the binding of ALW to anti-DNA antibodies by alanine scanning. Finally, ALW significantly inhibited the binding of murine and human lupus sera to dsDNA and glomeruli. In conclusion, by inhibiting the binding of polyclonal anti-DNA antibodies to autoantigens in vivo, the ALW peptide (or its derivatives) may potentially be a useful approach to block anti-DNA antibody binding to renal tissue. This article is protected by copyright. All rights reserved.
The Journal of biological chemistry, Jan 16, 2015
In recent years several groups have shown that isotype switching from IgM to IgG to IgA can affec... more In recent years several groups have shown that isotype switching from IgM to IgG to IgA can affect the affinity and specificity of antibodies (Abs) sharing identical variable (V) regions. However, whether the same applies to IgE is unknown. In this study we compared the fine specificity of V region identical IgE and IgA to Cryptococcus neoformans capsular polysaccharide and found that these differed in specificity from each other. The IgE and IgA paratopes were probed by nuclear magnetic resonance spectroscopy (NMR) with 15Nlabeled peptide mimetics of cryptococcal polysaccharide antigen (Ag). IgE was found to cleave the peptide at a much faster rate than V region-identical IgG subclasses and IgA, consistent with an altered paratope. Both IgE and IgA were opsonic for C. neoformans and protected against infection in mice. In summary, V region expression in the context of the epsilon constant (C) region results in specificity changes that are greater than observed for comparable IgG su...
Journal of biomolecular NMR, 2015
There has been a longstanding interest in being able to accurately predict NMR chemical shifts fr... more There has been a longstanding interest in being able to accurately predict NMR chemical shifts from structural data. Recent studies have focused on using molecular dynamics (MD) simulation data as input for improved prediction. Here we examine the accuracy of chemical shift prediction for intein systems, which have regions of intrinsic disorder. We find that using MD simulation data as input for chemical shift prediction does not consistently improve prediction accuracy over use of a static X-ray crystal structure. This appears to result from the complex conformational ensemble of the disordered protein segments. We show that using accelerated molecular dynamics (aMD) simulations improves chemical shift prediction, suggesting that methods which better sample the conformational ensemble like aMD are more appropriate tools for use in chemical shift prediction for proteins with disordered regions. Moreover, our study suggests that data accurately reflecting protein dynamics must be use...
Encyclopedia of Biophysics, 2013
Progress in Nuclear Magnetic Resonance Spectroscopy, 2010
Molecular Immunology, 2013
Until relatively recently the immunoglobulin molecule was viewed as composed of two independent d... more Until relatively recently the immunoglobulin molecule was viewed as composed of two independent domains comprised of the variable (V) and constant (C) regions. However, recent work has established that the C region mediates allosteric changes in the V region that can influence specificity and affinity. To further explore cross-domain interrelationship in murine IgG structure we carried out solution small angle X-ray scattering (SAXS) measurements for four V region identical IgG isotypes. SAXS analysis revealed elongated Y-shaped structures in solution with significantly different, isotype-dependent domain orientations. To further explore local C region effects on the V region, the IgG 3 Fab crystal structure from the same family was determined to 2.45Å resolution. The IgG 3 Fab crystal structure differs from a closely related previously solved IgG 1 Fab revealing significant structural differences, which may account for isotype-related specificity differences in V region identical Abs. Among the four murine isotypes, IgG 3 was the most different in solution with regards to overall structure as well as aggregate formation in solution suggesting that the greater apparent affinity of this isotype resulted from polyvalent complexes with enhanced avidity. Our results provide additional evidence that Ig V and C domains influence each other structurally and suggest that V region structure can have significant effects on overall Ig structure.
Journal of the American Chemical Society, 2013
Split inteins are a class of naturally occurring proteins that carry out protein splicing in tran... more Split inteins are a class of naturally occurring proteins that carry out protein splicing in trans. The chemical mechanism of protein trans-splicing is well-understood and has been exploited to develop several powerful protein engineering technologies. Split intein chemistry is preceded by efficient molecular recognition between two protomers that become intertwined in their bound state. It is currently unclear how this unique topology is achieved upon fragment association. Using biophysical techniques in conjunction with protein engineering methods, including segmental isotopic labeling, we show that one split intein fragment is partly folded, while the other is completely disordered. These polypeptides capture each other through their disordered regions and form an ordered intermediate with native-like structure at their interface. This intermediate then collapses into the canonical intein fold. This mechanism provides insight into the evolutionary constraints on split intein assembly and should enhance the development of split intein-based technologies.
Journal of the American Chemical Society, 2013
Split inteins play an important role in modern protein semisynthesis techniques. These naturally ... more Split inteins play an important role in modern protein semisynthesis techniques. These naturally occurring protein splicing domains can be used for in vitro and in vivo protein modification, peptide and protein cyclization, segmental isotopic labeling, and the construction of biosensors. The most well-characterized family of split inteins, the cyanobacterial DnaE inteins, show particular promise, as many of these can splice proteins in less than 1 min. Despite this fact, the activity of these inteins is context-dependent: certain peptide sequences surrounding their ligation junction (called local N-and Cexteins) are strongly preferred, while other sequences cause a dramatic reduction in the splicing kinetics and yield. These sequence constraints limit the utility of inteins, and thus, a more detailed understanding of their participation in protein splicing is needed. Here we present a thorough kinetic analysis of the relationship between C-extein composition and split intein activity. The results of these experiments were used to guide structural and molecular dynamics studies, which revealed that the motions of catalytic residues are constrained by the second C-extein residue, likely forcing them into an active conformation that promotes rapid protein splicing. Together, our structural and functional studies also highlight a key region of the intein structure that can be re-engineered to increase intein promiscuity.
![Research paper thumbnail of Erratum to “Structure and Dynamics of the P7 Protein from the Bacteriophage φ12” [J. Mol. Biol. (2008) 382, 402–422]](https://attachments.academia-assets.com/50483365/thumbnails/1.jpg)
](Journal of Molecular Biology, 2009
Journal of Molecular Biology, 2008
Cystoviruses are a class of enveloped double-stranded RNA viruses that use a multi-protein polyme... more Cystoviruses are a class of enveloped double-stranded RNA viruses that use a multi-protein polymerase complex (PX) to replicate and transcribe the viral genome. Though the structures of the polymerase and ATPase components of the cystoviral PX are known and their functional behavior understood to a large extent, no atomic-resolution structural information is available for the major capsid protein P1 that defines the overall structure and symmetry of the viral capsid and the essential protein P7. Towards obtaining a complete structural and functional understanding of the cystoviral PX, we have obtained the structure of P7 from the cystovirus ϕ12 at a resolution of 1.8 Å. The Nterminal core region (1-129) of P7 forms a novel homodimeric α/β-fold with structural similarities with BRCT domains implicated in multiple protein-protein interactions in DNA repair proteins. Our results combined with the known role of P7 in stabilizing the nucleation complex during capsid assembly hints towards its participation in key protein-protein interactions within the cystoviral PX. Additionally, we have found through solution NMR studies that the C-terminal tail of P7 (130-169) that is essential for virus viability, though highly disordered, contains a nascent helix. We demonstrate for the very first time, through NMR titrations, that P7 is capable of interacting with RNA. We find that both the N-terminal core and the dynamic C-terminus tail of P7 play a role in RNA recognition leading to a significant reduction of the degree of disorder in the C-terminal tail. Given the requirement of P7 in maintaining genome packaging efficiency and transcriptional fidelity, our data suggest a central biological role for P7/RNA interactions.
Journal of Biological Chemistry, 2014
Protein splicing is a posttranslational modification where intervening proteins (inteins) cleave ... more Protein splicing is a posttranslational modification where intervening proteins (inteins) cleave themselves from larger precursor proteins and ligate their flanking polypeptides (exteins) through a multistep chemical reaction. First thought to be an anomaly found in only a few organisms, protein splicing by inteins has since been observed in microorganisms from all domains of life. Despite this broad phylogenetic distribution, all inteins share common structural features such as a horseshoelike pseudo two-fold symmetric fold, several canonical sequence motifs, and similar splicing mechanisms. Intriguingly, the splicing efficiencies and substrate specificity of different inteins vary considerably, reflecting subtle changes in the chemical mechanism of splicing, linked to their local structure and dynamics. As intein chemistry has widespread use in protein chemistry, understanding the structural and dynamical aspects of inteins is crucial for intein engineering and the improvement of inteinbased technologies.
Journal of Biological Chemistry, 2012
The mechanism by which antibody constant region alters fine specificity is unknown. Results: Diff... more The mechanism by which antibody constant region alters fine specificity is unknown. Results: Different constant regions were found to change electronic and chemical properties of the antigen-binding site. Conclusion: Constant regions can affect the energy landscape of the variable region. Significance: These results are potentially critical for understanding fast, correct immune responses at the systems level and for future immunotherapy development.
Molecular Immunology, 2013
We previously demonstrated an important role of the constant region in the pathogenicity of anti-... more We previously demonstrated an important role of the constant region in the pathogenicity of anti-DNA antibodies. To determine the mechanisms by which the constant region affects autoantibody binding, a panel of isotype-switch variants (IgG1, IgG2a, IgG2b) was generated from the murine PL9-11 IgG3 autoantibody. The affinity of the PL9-11 antibody panel for histone was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine wavelength shifts of the antibody panel upon binding to DNA and histone. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure. SPR analysis revealed significant differences in histone binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the antibody isotype, while circular dichroism analysis determined that changes in antibody secondary structure content differed between isotypes upon antigen binding. Thus, the antigen binding affinity is dependent on the particular constant region expressed. Moreover, the effects of antibody binding to antigen were also constant region dependent. Alteration of secondary structures influenced by constant regions may explain differences in fine specificity of anti-DNA antibodies between antibodies with similar variable regions, as well as cross-reactivity of anti-DNA antibodies with non-DNA antigens.
J Mol Biol, 2008
Cystoviruses are a class of enveloped double-stranded RNA viruses that use a multiprotein polymer... more Cystoviruses are a class of enveloped double-stranded RNA viruses that use a multiprotein polymerase complex (PX) to replicate and transcribe the viral genome. Although the structures of the polymerase and ATPase components of the cystoviral PX are known and their functional behavior is understood to a large extent, no atomic-resolution structural information is available for the major capsid protein P1 that defines the overall structure and symmetry of the viral capsid and the essential protein P7. Toward obtaining a complete structural and functional understanding of the cystoviral PX, we have obtained the structure of P7 from the cystovirus ϕ12 at a resolution of 1.8 Å. The N-terminal core region (1-129) of P7 forms a novel homodimeric α/β-fold having structural similarities with BRCT domains implicated in multiple protein-protein interactions in DNA repair proteins. Our results, combined with the known role of P7 in stabilizing the nucleation complex during capsid assembly, hint toward its participation in key protein-protein interactions within the cystoviral PX. Additionally, we have found through solution NMR studies that the C-terminal tail of P7 (130-169) that is essential for virus viability, although highly disordered, contains a nascent helix. We demonstrate for the first time, through NMR titrations, that P7 is capable of interacting with RNA. We find that both the N-terminal core and the dynamic C-terminal tail of P7 play a role in RNA recognition. This interaction leads to a significant reduction of the degree of disorder in the C-terminal tail. Given the requirement of P7 in maintaining genome packaging efficiency and transcriptional fidelity, our data suggest a central biological role for P7-RNA interactions.
Journal of Molecular Biology, 2009
Molecular Immunology, 2016
Anti-DNA antibodies are the serological hallmark of systemic lupus erythematosus, and participate... more Anti-DNA antibodies are the serological hallmark of systemic lupus erythematosus, and participate in the pathogenesis of lupus nephritis by cross-reacting with multiple renal antigens. Previously, using a panel of murine anti-DNA IgGs that share identical variable regions but that differ in the constant regions, we demonstrated that the cross-reaction and renal pathogenicity of anti-DNA antibodies are isotype dependent. In this study, we investigated the catalytic potential of this anti-DNA antibody panel, and determined its isotype dependency. The three isotype switch variants (IgG1, IgG2a, IgG2b) and the parent IgG3 PL9-11 anti-DNA antibodies were compared in their catalysis of 500 base pair linear double stranded DNA and a 12-mer peptide (ALWPPNLHAWVP), by gel analysis, MALDI-TOF mass spectrometry, and nuclear magnetic resonance spectroscopy. The binding affinity of anti-DNA antibodies to double stranded DNA and peptide antigens were assessed by ELISA and surface plasmon resonance. We found that the PL9-11 antibody isotypes vary significantly in their potential to catalyze the cleavage of both linear and double stranded DNA and the proteolysis of peptides. The degree of the cleavage and proteolysis increases with the incubation temperature and time. While different PL9-11 isotypes have the same initial attack sites within the ALWPPNLHAWVP peptide, there was no correlation between binding affinity to the peptide and proteolysis rates. In conclusion, the catalytic properties of anti-DNA antibodies are isotype dependent. This finding provides further evidence that antibodies that share the same variable region, but which have different constant regions, are functionally distinct. The catalytic effects modulated by antibody constant regions need to be considered in the design of therapeutic antibodies (abzymes) and peptides designed to block pathogenic autoantibodies.
Clinical and experimental immunology, Jan 20, 2015
Anti-DNA antibodies play a pivotal role in the pathogenesis of lupus nephritis by cross reacting ... more Anti-DNA antibodies play a pivotal role in the pathogenesis of lupus nephritis by cross reacting with renal antigens. Previously, we demonstrated that the binding affinity of anti-DNA antibodies to self-antigens is isotype dependent. Furthermore, significant variability in renal pathogenicity was seen among a panel of anti-DNA isotypes (derived from a single murine IgG3 monoclonal antibody, PL9-11) that share identical variable regions. In this study, we sought to select peptide mimics that effectively inhibit the binding of all murine and human anti-DNA IgG isotypes to glomerular antigens. The PL9-11 panel of IgG anti-DNA antibodies (IgG1, IgG2a, IgG2b, and IgG3) was used for screening a 12-mer phage display library. Binding affinity was determined by surface plasmon resonance. ELISA, flow cytometry, and glomerular binding assays were used for the assessment of peptide inhibition of antibody binding to nuclear and kidney antigens. We identified a 12 amino acid peptide (ALWPPNLHAWVP...
Clinical & Experimental Immunology, 2015
Anti-DNA antibodies play a pivotal role in the pathogenesis of lupus nephritis by cross reacting ... more Anti-DNA antibodies play a pivotal role in the pathogenesis of lupus nephritis by cross reacting with renal antigens. Previously, we demonstrated that the binding affinity of anti-DNA antibodies to self-antigens is isotype dependent. Furthermore, significant variability in renal pathogenicity was seen among a panel of anti-DNA isotypes (derived from a single murine IgG3 monoclonal antibody, PL9-11) that share identical variable regions. In this study, we sought to select peptide mimics that effectively inhibit the binding of all murine and human anti-DNA IgG isotypes to glomerular antigens. The PL9-11 panel of IgG anti-DNA antibodies (IgG1, IgG2a, IgG2b, and IgG3) was used for screening a 12-mer phage display library. Binding affinity was determined by surface plasmon resonance. ELISA, flow cytometry, and glomerular binding assays were used for the assessment of peptide inhibition of antibody binding to nuclear and kidney antigens. We identified a 12 amino acid peptide (ALWPPNLHAWVP, or "ALW") which binds to all PL9-11 IgG isotypes. Pre-incubation with the ALW peptide significantly reduced the binding of the PL9-11 anti-DNA antibodies to DNA, laminin, mesangial cells, and isolated glomeruli. Furthermore, we confirmed the specificity of the amino acid sequence in the binding of ALW to anti-DNA antibodies by alanine scanning. Finally, ALW significantly inhibited the binding of murine and human lupus sera to dsDNA and glomeruli. In conclusion, by inhibiting the binding of polyclonal anti-DNA antibodies to autoantigens in vivo, the ALW peptide (or its derivatives) may potentially be a useful approach to block anti-DNA antibody binding to renal tissue. This article is protected by copyright. All rights reserved.
The Journal of biological chemistry, Jan 16, 2015
In recent years several groups have shown that isotype switching from IgM to IgG to IgA can affec... more In recent years several groups have shown that isotype switching from IgM to IgG to IgA can affect the affinity and specificity of antibodies (Abs) sharing identical variable (V) regions. However, whether the same applies to IgE is unknown. In this study we compared the fine specificity of V region identical IgE and IgA to Cryptococcus neoformans capsular polysaccharide and found that these differed in specificity from each other. The IgE and IgA paratopes were probed by nuclear magnetic resonance spectroscopy (NMR) with 15Nlabeled peptide mimetics of cryptococcal polysaccharide antigen (Ag). IgE was found to cleave the peptide at a much faster rate than V region-identical IgG subclasses and IgA, consistent with an altered paratope. Both IgE and IgA were opsonic for C. neoformans and protected against infection in mice. In summary, V region expression in the context of the epsilon constant (C) region results in specificity changes that are greater than observed for comparable IgG su...
Journal of biomolecular NMR, 2015
There has been a longstanding interest in being able to accurately predict NMR chemical shifts fr... more There has been a longstanding interest in being able to accurately predict NMR chemical shifts from structural data. Recent studies have focused on using molecular dynamics (MD) simulation data as input for improved prediction. Here we examine the accuracy of chemical shift prediction for intein systems, which have regions of intrinsic disorder. We find that using MD simulation data as input for chemical shift prediction does not consistently improve prediction accuracy over use of a static X-ray crystal structure. This appears to result from the complex conformational ensemble of the disordered protein segments. We show that using accelerated molecular dynamics (aMD) simulations improves chemical shift prediction, suggesting that methods which better sample the conformational ensemble like aMD are more appropriate tools for use in chemical shift prediction for proteins with disordered regions. Moreover, our study suggests that data accurately reflecting protein dynamics must be use...
Encyclopedia of Biophysics, 2013
Progress in Nuclear Magnetic Resonance Spectroscopy, 2010
Molecular Immunology, 2013
Until relatively recently the immunoglobulin molecule was viewed as composed of two independent d... more Until relatively recently the immunoglobulin molecule was viewed as composed of two independent domains comprised of the variable (V) and constant (C) regions. However, recent work has established that the C region mediates allosteric changes in the V region that can influence specificity and affinity. To further explore cross-domain interrelationship in murine IgG structure we carried out solution small angle X-ray scattering (SAXS) measurements for four V region identical IgG isotypes. SAXS analysis revealed elongated Y-shaped structures in solution with significantly different, isotype-dependent domain orientations. To further explore local C region effects on the V region, the IgG 3 Fab crystal structure from the same family was determined to 2.45Å resolution. The IgG 3 Fab crystal structure differs from a closely related previously solved IgG 1 Fab revealing significant structural differences, which may account for isotype-related specificity differences in V region identical Abs. Among the four murine isotypes, IgG 3 was the most different in solution with regards to overall structure as well as aggregate formation in solution suggesting that the greater apparent affinity of this isotype resulted from polyvalent complexes with enhanced avidity. Our results provide additional evidence that Ig V and C domains influence each other structurally and suggest that V region structure can have significant effects on overall Ig structure.
Journal of the American Chemical Society, 2013
Split inteins are a class of naturally occurring proteins that carry out protein splicing in tran... more Split inteins are a class of naturally occurring proteins that carry out protein splicing in trans. The chemical mechanism of protein trans-splicing is well-understood and has been exploited to develop several powerful protein engineering technologies. Split intein chemistry is preceded by efficient molecular recognition between two protomers that become intertwined in their bound state. It is currently unclear how this unique topology is achieved upon fragment association. Using biophysical techniques in conjunction with protein engineering methods, including segmental isotopic labeling, we show that one split intein fragment is partly folded, while the other is completely disordered. These polypeptides capture each other through their disordered regions and form an ordered intermediate with native-like structure at their interface. This intermediate then collapses into the canonical intein fold. This mechanism provides insight into the evolutionary constraints on split intein assembly and should enhance the development of split intein-based technologies.
Journal of the American Chemical Society, 2013
Split inteins play an important role in modern protein semisynthesis techniques. These naturally ... more Split inteins play an important role in modern protein semisynthesis techniques. These naturally occurring protein splicing domains can be used for in vitro and in vivo protein modification, peptide and protein cyclization, segmental isotopic labeling, and the construction of biosensors. The most well-characterized family of split inteins, the cyanobacterial DnaE inteins, show particular promise, as many of these can splice proteins in less than 1 min. Despite this fact, the activity of these inteins is context-dependent: certain peptide sequences surrounding their ligation junction (called local N-and Cexteins) are strongly preferred, while other sequences cause a dramatic reduction in the splicing kinetics and yield. These sequence constraints limit the utility of inteins, and thus, a more detailed understanding of their participation in protein splicing is needed. Here we present a thorough kinetic analysis of the relationship between C-extein composition and split intein activity. The results of these experiments were used to guide structural and molecular dynamics studies, which revealed that the motions of catalytic residues are constrained by the second C-extein residue, likely forcing them into an active conformation that promotes rapid protein splicing. Together, our structural and functional studies also highlight a key region of the intein structure that can be re-engineered to increase intein promiscuity.
Journal of Molecular Biology, 2009
Journal of Molecular Biology, 2008
Cystoviruses are a class of enveloped double-stranded RNA viruses that use a multi-protein polyme... more Cystoviruses are a class of enveloped double-stranded RNA viruses that use a multi-protein polymerase complex (PX) to replicate and transcribe the viral genome. Though the structures of the polymerase and ATPase components of the cystoviral PX are known and their functional behavior understood to a large extent, no atomic-resolution structural information is available for the major capsid protein P1 that defines the overall structure and symmetry of the viral capsid and the essential protein P7. Towards obtaining a complete structural and functional understanding of the cystoviral PX, we have obtained the structure of P7 from the cystovirus ϕ12 at a resolution of 1.8 Å. The Nterminal core region (1-129) of P7 forms a novel homodimeric α/β-fold with structural similarities with BRCT domains implicated in multiple protein-protein interactions in DNA repair proteins. Our results combined with the known role of P7 in stabilizing the nucleation complex during capsid assembly hints towards its participation in key protein-protein interactions within the cystoviral PX. Additionally, we have found through solution NMR studies that the C-terminal tail of P7 (130-169) that is essential for virus viability, though highly disordered, contains a nascent helix. We demonstrate for the very first time, through NMR titrations, that P7 is capable of interacting with RNA. We find that both the N-terminal core and the dynamic C-terminus tail of P7 play a role in RNA recognition leading to a significant reduction of the degree of disorder in the C-terminal tail. Given the requirement of P7 in maintaining genome packaging efficiency and transcriptional fidelity, our data suggest a central biological role for P7/RNA interactions.
Journal of Biological Chemistry, 2014
Protein splicing is a posttranslational modification where intervening proteins (inteins) cleave ... more Protein splicing is a posttranslational modification where intervening proteins (inteins) cleave themselves from larger precursor proteins and ligate their flanking polypeptides (exteins) through a multistep chemical reaction. First thought to be an anomaly found in only a few organisms, protein splicing by inteins has since been observed in microorganisms from all domains of life. Despite this broad phylogenetic distribution, all inteins share common structural features such as a horseshoelike pseudo two-fold symmetric fold, several canonical sequence motifs, and similar splicing mechanisms. Intriguingly, the splicing efficiencies and substrate specificity of different inteins vary considerably, reflecting subtle changes in the chemical mechanism of splicing, linked to their local structure and dynamics. As intein chemistry has widespread use in protein chemistry, understanding the structural and dynamical aspects of inteins is crucial for intein engineering and the improvement of inteinbased technologies.
Journal of Biological Chemistry, 2012
The mechanism by which antibody constant region alters fine specificity is unknown. Results: Diff... more The mechanism by which antibody constant region alters fine specificity is unknown. Results: Different constant regions were found to change electronic and chemical properties of the antigen-binding site. Conclusion: Constant regions can affect the energy landscape of the variable region. Significance: These results are potentially critical for understanding fast, correct immune responses at the systems level and for future immunotherapy development.
Molecular Immunology, 2013
We previously demonstrated an important role of the constant region in the pathogenicity of anti-... more We previously demonstrated an important role of the constant region in the pathogenicity of anti-DNA antibodies. To determine the mechanisms by which the constant region affects autoantibody binding, a panel of isotype-switch variants (IgG1, IgG2a, IgG2b) was generated from the murine PL9-11 IgG3 autoantibody. The affinity of the PL9-11 antibody panel for histone was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine wavelength shifts of the antibody panel upon binding to DNA and histone. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure. SPR analysis revealed significant differences in histone binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the antibody isotype, while circular dichroism analysis determined that changes in antibody secondary structure content differed between isotypes upon antigen binding. Thus, the antigen binding affinity is dependent on the particular constant region expressed. Moreover, the effects of antibody binding to antigen were also constant region dependent. Alteration of secondary structures influenced by constant regions may explain differences in fine specificity of anti-DNA antibodies between antibodies with similar variable regions, as well as cross-reactivity of anti-DNA antibodies with non-DNA antigens.