Journal Articles
Deformed grids for single-particle cryo-electron microscopy of specimens exhibiting a preferred orientation
[Apr 2013]
Publication date: Available online 26 March 2013
Source:Journal of Structural Biology
Author(s): Ying Liu , Xing Meng , Zheng Liu
For biological samples showing a preferred orientation on the carbon support film of an electron microscope (EM) grid, accurate three-dimensional (3D) reconstructions by single-particle cryo-EM require data collection in which the specimen grids are tilted in the microscope, to obtain adequate numbers of particles that cover the high-degree angular distribution. However, image drift caused by the electron beam interacting with the cryo specimen becomes severe when grids are tilted to high angles (>30°). We produced deformed grids by applying a deliberate mechanical deformation to EM grids containing a thin carbon film supported by a thick holey carbon film. We applied cryo-EM using deformed grids to the isolated cardiac ryanodine receptor, an ion channel complex known to assume a preferred orientation on the carbon support film. These grids contained more particles having high Euler angle orientations without the need to tilt the specimen grids. Meanwhile, the drifting that was apparent in the images was reduced from that typical of images from tilted regular EM grids. This was achieved by imaging particles in holes close to the deformed areas, where carbon films were locally bent, offering planes of inclination with various angles. The deformed grids improve the efficiency and quality of data collection for single-particle cryo-EM of samples showing a limited range of orientations.
Source:Journal of Structural Biology
Author(s): Ying Liu , Xing Meng , Zheng Liu
For biological samples showing a preferred orientation on the carbon support film of an electron microscope (EM) grid, accurate three-dimensional (3D) reconstructions by single-particle cryo-EM require data collection in which the specimen grids are tilted in the microscope, to obtain adequate numbers of particles that cover the high-degree angular distribution. However, image drift caused by the electron beam interacting with the cryo specimen becomes severe when grids are tilted to high angles (>30°). We produced deformed grids by applying a deliberate mechanical deformation to EM grids containing a thin carbon film supported by a thick holey carbon film. We applied cryo-EM using deformed grids to the isolated cardiac ryanodine receptor, an ion channel complex known to assume a preferred orientation on the carbon support film. These grids contained more particles having high Euler angle orientations without the need to tilt the specimen grids. Meanwhile, the drifting that was apparent in the images was reduced from that typical of images from tilted regular EM grids. This was achieved by imaging particles in holes close to the deformed areas, where carbon films were locally bent, offering planes of inclination with various angles. The deformed grids improve the efficiency and quality of data collection for single-particle cryo-EM of samples showing a limited range of orientations.
Categories: Journal Articles
Affinity maturation of a computationally designed binding protein affords a functional but disordered polypeptide
[Apr 2013]
Publication date: Available online 26 March 2013
Source:Journal of Structural Biology
Author(s): Maren Butz , Peter Kast , Donald Hilvert
Computational methods have been recently applied to the design of protein–protein interfaces. Using this approach, a 61 amino acid long protein called Spider Roll was engineered to recognize the kinase domain of the human p21-activated kinase 1 (PAK1) with good specificity but modest affinity (K D =100μM). Here we show that this artificial protein can be optimized by yeast surface display and fluorescence-activated cell sorting. After three rounds of mutagenesis and screening, a diverse set of tighter binding variants was obtained. A representative binder, MSR7, has a >102-fold higher affinity for PAK1 when displayed on yeast and a 6 to 11-fold advantage when produced free in solution. In contrast to the starting Spider Roll protein, however, MSR7 unexpectedly exhibits characteristics typical of partially disordered proteins, including lower α-helical content, non-cooperative thermal denaturation, and NMR data showing peak broadening and poor signal dispersion. Although conformational disorder is increasingly recognized as an important property of proteins involved in cellular signaling and regulation, it is poorly modeled by current computational methods. Explicit consideration of structural flexibility may improve future protein designs and provide deeper insight into molecular events at protein–protein interfaces.
Source:Journal of Structural Biology
Author(s): Maren Butz , Peter Kast , Donald Hilvert
Computational methods have been recently applied to the design of protein–protein interfaces. Using this approach, a 61 amino acid long protein called Spider Roll was engineered to recognize the kinase domain of the human p21-activated kinase 1 (PAK1) with good specificity but modest affinity (K D =100μM). Here we show that this artificial protein can be optimized by yeast surface display and fluorescence-activated cell sorting. After three rounds of mutagenesis and screening, a diverse set of tighter binding variants was obtained. A representative binder, MSR7, has a >102-fold higher affinity for PAK1 when displayed on yeast and a 6 to 11-fold advantage when produced free in solution. In contrast to the starting Spider Roll protein, however, MSR7 unexpectedly exhibits characteristics typical of partially disordered proteins, including lower α-helical content, non-cooperative thermal denaturation, and NMR data showing peak broadening and poor signal dispersion. Although conformational disorder is increasingly recognized as an important property of proteins involved in cellular signaling and regulation, it is poorly modeled by current computational methods. Explicit consideration of structural flexibility may improve future protein designs and provide deeper insight into molecular events at protein–protein interfaces.
Categories: Journal Articles
Correlative microscopy methods that maximize specimen fidelity and data completeness, and improve molecular localization capabilities
[Apr 2013]
Publication date: Available online 24 March 2013
Source:Journal of Structural Biology
Author(s): Elizabeth A. Smith , Bertrand P. Cinquin , Gerry McDermott , Mark A. Le Gros , Dilworth Y. Parkinson , Hong Tae Kim , Carolyn A. Larabell
Correlative microscopy techniques interrogate biological systems more thoroughly than is possible using a single modality. This is particularly true if disparate data types can be acquired from the same specimen. Recently, there has been significant progress towards combining the structural information obtained from soft X-ray tomography (SXT) with molecular localization data. Here we will compare methods for determining the position of molecules in a cell viewed by SXT, including direct visualization using electron dense labels, and by indirect methods, such as fluorescence microscopy and high numerical aperture cryo-light microscopy. We will also discuss available options for preserving the in vivo structure and organization of the specimen during multi-modal data collection, and how some simple specimen mounting concepts can ensure maximal data completeness in correlative imaging experiments.
Source:Journal of Structural Biology
Author(s): Elizabeth A. Smith , Bertrand P. Cinquin , Gerry McDermott , Mark A. Le Gros , Dilworth Y. Parkinson , Hong Tae Kim , Carolyn A. Larabell
Correlative microscopy techniques interrogate biological systems more thoroughly than is possible using a single modality. This is particularly true if disparate data types can be acquired from the same specimen. Recently, there has been significant progress towards combining the structural information obtained from soft X-ray tomography (SXT) with molecular localization data. Here we will compare methods for determining the position of molecules in a cell viewed by SXT, including direct visualization using electron dense labels, and by indirect methods, such as fluorescence microscopy and high numerical aperture cryo-light microscopy. We will also discuss available options for preserving the in vivo structure and organization of the specimen during multi-modal data collection, and how some simple specimen mounting concepts can ensure maximal data completeness in correlative imaging experiments.
Categories: Journal Articles
The human ITPA polymorphic variant P32T is destabilized by the unpacking of the hydrophobic core
[Apr 2013]
Publication date: Available online 23 March 2013
Source:Journal of Structural Biology
Author(s): Peter D. Simone , Lucas R. Struble , Admir Kellezi , Carrie A. Brown , Corinn E. Grabow , Irine Khutsishvili , Luis A. Marky , Youri I. Pavlov , Gloria E.O. Borgstahl
Inosine triphosphate pyrophosphatase (ITPA), a key enzyme involved in maintaining the purity of cellular nucleoside triphosphate pools, specifically recognizes inosine triphosphate and xanthosine triphosphate (including the deoxyribose forms) and detoxifies them by catalyzing the hydrolysis of a phosphoanhydride bond, releasing pyrophosphate. This prevents their inappropriate use as substrates in enzymatic reactions utilizing (d)ATP or (d)GTP. A human genetic polymorphism leads to the substitution of Thr for Pro32 (P32T) and causes ITPA deficiency in erythrocytes, with heterozygotes having on average 22.5% residual activity, and homozygotes having undetectable activity. This polymorphism has been implicated in modulating patients’ response to mercaptopurines and ribavirin. Human fibroblasts containing this variant have elevated genomic instability upon treatment with base analogs. We find that the wild-type and P32T forms are dimeric in solution and in the crystal structure. This abolishes the previous speculation that the P32T change disrupts dimerization as a mechanism of inactivation. The only difference in structure from the wild-type protein is that the area surrounding Thr32 is disrupted. Phe31 is flipped from the hydrophobic core out into the solvent, leaving a hole in the hydrophobic core of the protein which likely accounts for the reduced thermal stability of P32T ITPA and ultimately leads to its susceptibility to degradation in human cells. Circular dichroism and thermal denaturation studies confirm these structural results. We propose that the dimer of P32T variant subunit with wild-type subunit is degraded in cells similarly to the P32T homodimer explaining the level of loss of ITPA activity in heterozygotes.
Source:Journal of Structural Biology
Author(s): Peter D. Simone , Lucas R. Struble , Admir Kellezi , Carrie A. Brown , Corinn E. Grabow , Irine Khutsishvili , Luis A. Marky , Youri I. Pavlov , Gloria E.O. Borgstahl
Inosine triphosphate pyrophosphatase (ITPA), a key enzyme involved in maintaining the purity of cellular nucleoside triphosphate pools, specifically recognizes inosine triphosphate and xanthosine triphosphate (including the deoxyribose forms) and detoxifies them by catalyzing the hydrolysis of a phosphoanhydride bond, releasing pyrophosphate. This prevents their inappropriate use as substrates in enzymatic reactions utilizing (d)ATP or (d)GTP. A human genetic polymorphism leads to the substitution of Thr for Pro32 (P32T) and causes ITPA deficiency in erythrocytes, with heterozygotes having on average 22.5% residual activity, and homozygotes having undetectable activity. This polymorphism has been implicated in modulating patients’ response to mercaptopurines and ribavirin. Human fibroblasts containing this variant have elevated genomic instability upon treatment with base analogs. We find that the wild-type and P32T forms are dimeric in solution and in the crystal structure. This abolishes the previous speculation that the P32T change disrupts dimerization as a mechanism of inactivation. The only difference in structure from the wild-type protein is that the area surrounding Thr32 is disrupted. Phe31 is flipped from the hydrophobic core out into the solvent, leaving a hole in the hydrophobic core of the protein which likely accounts for the reduced thermal stability of P32T ITPA and ultimately leads to its susceptibility to degradation in human cells. Circular dichroism and thermal denaturation studies confirm these structural results. We propose that the dimer of P32T variant subunit with wild-type subunit is degraded in cells similarly to the P32T homodimer explaining the level of loss of ITPA activity in heterozygotes.
Categories: Journal Articles
Fast and accurate reference-free alignment of subtomograms
[Apr 2013]
Publication date: Available online 22 March 2013
Source:Journal of Structural Biology
Author(s): Yuxiang Chen , Stefan Pfeffer , Thomas Hrabe , Jan Michael Schuller , Friedrich Förster
In cryoelectron tomography alignment and averaging of subtomograms, each dnepicting the same macromolecule, improves the resolution compared to the individual subtomogram. Major challenges of subtomogram alignment are noise enhancement due to overfitting, the bias of an initial reference in the iterative alignment process, and the computational cost of processing increasingly large amounts of data. Here, we propose an efficient and accurate alignment algorithm via a generalized convolution theorem, which allows computation of a constrained correlation function using spherical harmonics. This formulation increases computational speed of rotational matching dramatically compared to rotation search in Cartesian space without sacrificing accuracy in contrast to other spherical harmonic based approaches. Using this sampling method, a reference-free alignment procedure is proposed to tackle reference bias and overfitting, which also includes contrast transfer function correction by Wiener filtering. Application of the method to simulated data allowed us to obtain resolutions near the ground truth. For two experimental datasets, ribosomes from yeast lysate and purified 20S proteasomes, we achieved reconstructions of approximately 20Å and 16Å, respectively. The software is ready-to-use and made public to the community.
Source:Journal of Structural Biology
Author(s): Yuxiang Chen , Stefan Pfeffer , Thomas Hrabe , Jan Michael Schuller , Friedrich Förster
In cryoelectron tomography alignment and averaging of subtomograms, each dnepicting the same macromolecule, improves the resolution compared to the individual subtomogram. Major challenges of subtomogram alignment are noise enhancement due to overfitting, the bias of an initial reference in the iterative alignment process, and the computational cost of processing increasingly large amounts of data. Here, we propose an efficient and accurate alignment algorithm via a generalized convolution theorem, which allows computation of a constrained correlation function using spherical harmonics. This formulation increases computational speed of rotational matching dramatically compared to rotation search in Cartesian space without sacrificing accuracy in contrast to other spherical harmonic based approaches. Using this sampling method, a reference-free alignment procedure is proposed to tackle reference bias and overfitting, which also includes contrast transfer function correction by Wiener filtering. Application of the method to simulated data allowed us to obtain resolutions near the ground truth. For two experimental datasets, ribosomes from yeast lysate and purified 20S proteasomes, we achieved reconstructions of approximately 20Å and 16Å, respectively. The software is ready-to-use and made public to the community.
Categories: Journal Articles
Interactions of the α-subunits of heterotrimeric G-proteins with GPCRs, effectors and RGS proteins: A critical review and analysis of interacting surfaces, conformational shifts, structural diversity and electrostatic potentials
[Apr 2013]
Publication date: Available online 21 March 2013
Source:Journal of Structural Biology
Author(s): Fotis A. Baltoumas , Margarita C. Theodoropoulou , Stavros J. Hamodrakas
G-protein coupled receptors (GPCRs) are one of the largest families of membrane receptors in eukaryotes. Heterotrimeric G-proteins, composed of α, β and γ subunits, are important molecular switches in the mediation of GPCR signaling. Receptor stimulation after the binding of a suitable ligand leads to G-protein heterotrimer activation and dissociation into the Gα subunit and Gβγ heterodimer. These subunits then interact with a large number of effectors, leading to several cell responses. We studied the interactions between Gα subunits and their binding partners, using information from structural, mutagenesis and Bioinformatics studies, and conducted a series of comparisons of sequence, structure, electrostatic properties and intermolecular energies among different Gα families and subfamilies. We identified a number of Gα surfaces that may, in several occasions, participate in interactions with receptors as well as effectors. The study of Gα interacting surfaces in terms of sequence, structure and electrostatic potential reveals features that may account for the Gα subunit’s behavior towards its interacting partners. The electrostatic properties of the Gα subunits, which in some cases differ greatly not only between families but also between subfamilies, as well as the G-protein interacting surfaces of effectors and regulators of G-protein signaling (RGS) suggest that electrostatic complementarity may be an important factor in G-protein interactions. Energy calculations also support this notion. This information may be useful in future studies of G-protein interactions with GPCRs and effectors.
Source:Journal of Structural Biology
Author(s): Fotis A. Baltoumas , Margarita C. Theodoropoulou , Stavros J. Hamodrakas
G-protein coupled receptors (GPCRs) are one of the largest families of membrane receptors in eukaryotes. Heterotrimeric G-proteins, composed of α, β and γ subunits, are important molecular switches in the mediation of GPCR signaling. Receptor stimulation after the binding of a suitable ligand leads to G-protein heterotrimer activation and dissociation into the Gα subunit and Gβγ heterodimer. These subunits then interact with a large number of effectors, leading to several cell responses. We studied the interactions between Gα subunits and their binding partners, using information from structural, mutagenesis and Bioinformatics studies, and conducted a series of comparisons of sequence, structure, electrostatic properties and intermolecular energies among different Gα families and subfamilies. We identified a number of Gα surfaces that may, in several occasions, participate in interactions with receptors as well as effectors. The study of Gα interacting surfaces in terms of sequence, structure and electrostatic potential reveals features that may account for the Gα subunit’s behavior towards its interacting partners. The electrostatic properties of the Gα subunits, which in some cases differ greatly not only between families but also between subfamilies, as well as the G-protein interacting surfaces of effectors and regulators of G-protein signaling (RGS) suggest that electrostatic complementarity may be an important factor in G-protein interactions. Energy calculations also support this notion. This information may be useful in future studies of G-protein interactions with GPCRs and effectors.
Categories: Journal Articles
Hysteresis in swelling and in sorption of wood tissue
[Apr 2013]
Publication date: Available online 21 March 2013
Source:Journal of Structural Biology
Author(s): Alessandra Patera , Dominique Derome , Michele Griffa , Jan Carmeliet
The swelling and shrinkage of four Picea abies (L. Karst) wood tissue homogeneous samples, of porosity varying between 45% and 78%, is documented with high-resolution synchrotron radiation phase-contrast X-ray tomographic microscopy. We report measurements of the reversible moisture-induced orthotropic swelling/shrinkage strains. Hysteresis is observed when the swelling/shrinkage strain is considered as a function of relative humidity, except for the very high porosity sample. Hysteresis is no longer present when swelling/shrinkage strains are considered versus moisture content, indicating that wood deforms to the same extent whether an amount of moisture is desorbed or adsorbed. Furthermore, swelling anisotropy, in the tangential and radial directions, is found to increase with increasing porosity. The most homogeneous behaviour for a group of cells is found for 30–50 cells, smaller/larger groups having higher orders of variations.
Source:Journal of Structural Biology
Author(s): Alessandra Patera , Dominique Derome , Michele Griffa , Jan Carmeliet
The swelling and shrinkage of four Picea abies (L. Karst) wood tissue homogeneous samples, of porosity varying between 45% and 78%, is documented with high-resolution synchrotron radiation phase-contrast X-ray tomographic microscopy. We report measurements of the reversible moisture-induced orthotropic swelling/shrinkage strains. Hysteresis is observed when the swelling/shrinkage strain is considered as a function of relative humidity, except for the very high porosity sample. Hysteresis is no longer present when swelling/shrinkage strains are considered versus moisture content, indicating that wood deforms to the same extent whether an amount of moisture is desorbed or adsorbed. Furthermore, swelling anisotropy, in the tangential and radial directions, is found to increase with increasing porosity. The most homogeneous behaviour for a group of cells is found for 30–50 cells, smaller/larger groups having higher orders of variations.
Categories: Journal Articles
Linking receptor activation to changes in Sw I and II of Gα proteins
[Apr 2013]
Publication date: Available online 4 March 2013
Source:Journal of Structural Biology
Author(s): Heidi E. Hamm , Ali I. Kaya , James A. Gilbert III , Anita M. Preininger
G-protein coupled receptors catalyze nucleotide exchange on G proteins, which results in subunit dissociation and effector activation. In the recent β2AR-Gs structure, portions of Switch I and II of Gα are not fully elucidated. We paired fluorescence studies of receptor-Gαi interactions with the β2AR-Gs and other Gi structures to investigate changes in Switch I and II during receptor activation and GTP binding. The β2/β3 loop containing Leu194 of Gαi is located between Switches I and II, in close proximity to IC2 of the receptor and the C-terminus of Gα, thus providing an allosteric connection between these Switches and receptor activation. We compared the environment of residues in myristoylated Gαi proteins in the heterotrimer to that upon receptor activation and subsequent GTP binding. Upon receptor activation, residues in both Switch regions are less solvent-exposed, as compared to the heterotrimer. Upon GTPγS binding, the environment of several residues in Switch I resemble the receptor-bound state, while Switch II residues display effects on their environment which are consistent with their role in GTP binding and Gβγ dissociation. The ability to merge available crystal structures with solution studies is a powerful tool to gain insight into conformational changes associated with receptor-mediated Gi protein activation.
Source:Journal of Structural Biology
Author(s): Heidi E. Hamm , Ali I. Kaya , James A. Gilbert III , Anita M. Preininger
G-protein coupled receptors catalyze nucleotide exchange on G proteins, which results in subunit dissociation and effector activation. In the recent β2AR-Gs structure, portions of Switch I and II of Gα are not fully elucidated. We paired fluorescence studies of receptor-Gαi interactions with the β2AR-Gs and other Gi structures to investigate changes in Switch I and II during receptor activation and GTP binding. The β2/β3 loop containing Leu194 of Gαi is located between Switches I and II, in close proximity to IC2 of the receptor and the C-terminus of Gα, thus providing an allosteric connection between these Switches and receptor activation. We compared the environment of residues in myristoylated Gαi proteins in the heterotrimer to that upon receptor activation and subsequent GTP binding. Upon receptor activation, residues in both Switch regions are less solvent-exposed, as compared to the heterotrimer. Upon GTPγS binding, the environment of several residues in Switch I resemble the receptor-bound state, while Switch II residues display effects on their environment which are consistent with their role in GTP binding and Gβγ dissociation. The ability to merge available crystal structures with solution studies is a powerful tool to gain insight into conformational changes associated with receptor-mediated Gi protein activation.
Categories: Journal Articles
Cover 2 - Editorial Board
[Apr 2013]
Publication date: March 2013
Source:Journal of Structural Biology, Volume 181, Issue 3
Source:Journal of Structural Biology, Volume 181, Issue 3
Categories: Journal Articles
Table of Contents / barcode
[Apr 2013]
Publication date: March 2013
Source:Journal of Structural Biology, Volume 181, Issue 3
Source:Journal of Structural Biology, Volume 181, Issue 3
Categories: Journal Articles
Editorial Board
[Apr 2013]
Publication date: 13 May 2013
Source:Journal of Molecular Biology, Volume 425, Issue 9
Source:Journal of Molecular Biology, Volume 425, Issue 9
Categories: Journal Articles
Contents
[Apr 2013]
Publication date: 13 May 2013
Source:Journal of Molecular Biology, Volume 425, Issue 9
Source:Journal of Molecular Biology, Volume 425, Issue 9
Categories: Journal Articles
Allosteric Interactions after 50Years
[Apr 2013]
Publication date: 13 May 2013
Source:Journal of Molecular Biology, Volume 425, Issue 9
Author(s): Stuart J. Edelstein
Source:Journal of Molecular Biology, Volume 425, Issue 9
Author(s): Stuart J. Edelstein
Categories: Journal Articles
The Origins of Allostery: From Personal Memories to Material for the Future
[Apr 2013]
Publication date: 13 May 2013
Source:Journal of Molecular Biology, Volume 425, Issue 9
Author(s): Jean-Pierre Changeux
This introductory text evokes personal memories about the origins and definition of the word allosteric that is meant to qualify an “indirect” interaction between topographically and stereospecifically distinct sites, mediated by a discrete and reversible conformational change of the protein. The allosteric transition paradigm adds a new dimension and creates new openings to understanding receptors, signal transduction and drug design.
Graphical abstract Highlights ► The mechanism of feedback inhibition and the origins of the word “allosteric” (1961). ► The concept of allosteric transition and Koshland induced fit (1963). ► The MWC (Monod–Wyman–Changeux) model (1965) and its experimental test. ► Its extension to membrane assemblies: a subunit-based general model for cooperative interactions [Changeux, J. -P., Thiéry, J. -P., Tung, T. & Kittel, C. (1967). On the cooperativity of biological membranes. Proc. Natl. Acad. Sci. USA 57, 335–341]. ► The nicotinic acetylcholine receptor, allosteric modulation and drug design [Changeux, J. -P., Kasai, M. & Lee, C. Y. (1970). Use of a snake venom toxin to characterize the cholinergic receptor protein. Proc. Natl. Acad. Sci. USA 67, 1241–1247].
Source:Journal of Molecular Biology, Volume 425, Issue 9
Author(s): Jean-Pierre Changeux
This introductory text evokes personal memories about the origins and definition of the word allosteric that is meant to qualify an “indirect” interaction between topographically and stereospecifically distinct sites, mediated by a discrete and reversible conformational change of the protein. The allosteric transition paradigm adds a new dimension and creates new openings to understanding receptors, signal transduction and drug design.
Graphical abstract Highlights ► The mechanism of feedback inhibition and the origins of the word “allosteric” (1961). ► The concept of allosteric transition and Koshland induced fit (1963). ► The MWC (Monod–Wyman–Changeux) model (1965) and its experimental test. ► Its extension to membrane assemblies: a subunit-based general model for cooperative interactions [Changeux, J. -P., Thiéry, J. -P., Tung, T. & Kittel, C. (1967). On the cooperativity of biological membranes. Proc. Natl. Acad. Sci. USA 57, 335–341]. ► The nicotinic acetylcholine receptor, allosteric modulation and drug design [Changeux, J. -P., Kasai, M. & Lee, C. Y. (1970). Use of a snake venom toxin to characterize the cholinergic receptor protein. Proc. Natl. Acad. Sci. USA 67, 1241–1247].
Categories: Journal Articles
The Design of an Enzyme: A Chronology on the Controversy
[Apr 2013]
Publication date: 13 May 2013
Source:Journal of Molecular Biology, Volume 425, Issue 9
Author(s): Henri Buc
After the publication of the Monod–Wyman–Changeux model, a controversy arose between Jacques Monod, Francis Crick and Jeffries Wyman about the comparison of the regulatory performances of an oligomer undergoing a concerted transition between two states and a monomer having the same composition and subjected to a similar conformational equilibrium. The controversy took place between September 1965 and March 1966. It gave rise to several unpublished notes. Numerous misunderstandings between the participants were not fully dissipated as the controversy abruptly ended.
Graphical abstract Highlights ► The manuscript entitled, “A Footnote on Allostery”, rediscovered by Stuart Edelstein, is replaced within its historical context. ► Misunderstandings that arose among Monod, Crick and Wyman after the publication of the MWC (Monod–Wyman–Changeux) model are reviewed. ► Crick and Wyman provided a correct thermodynamic treatment but with the use of an ill-defined entity, the reference monomer.
Source:Journal of Molecular Biology, Volume 425, Issue 9
Author(s): Henri Buc
After the publication of the Monod–Wyman–Changeux model, a controversy arose between Jacques Monod, Francis Crick and Jeffries Wyman about the comparison of the regulatory performances of an oligomer undergoing a concerted transition between two states and a monomer having the same composition and subjected to a similar conformational equilibrium. The controversy took place between September 1965 and March 1966. It gave rise to several unpublished notes. Numerous misunderstandings between the participants were not fully dissipated as the controversy abruptly ended.
Graphical abstract Highlights ► The manuscript entitled, “A Footnote on Allostery”, rediscovered by Stuart Edelstein, is replaced within its historical context. ► Misunderstandings that arose among Monod, Crick and Wyman after the publication of the MWC (Monod–Wyman–Changeux) model are reviewed. ► Crick and Wyman provided a correct thermodynamic treatment but with the use of an ill-defined entity, the reference monomer.
Categories: Journal Articles
The Propagation of Allosteric States in Large Multiprotein Complexes
[Apr 2013]
Publication date: 13 May 2013
Source:Journal of Molecular Biology, Volume 425, Issue 9
Author(s): Dennis Bray
A statistical view of allostery leads to a more nuanced and physically realistic picture of protein cooperativity. If the conformational state of one protein molecule in a multiprotein complex influences the probability of a particular conformation in a neighbouring protein, then changes can propagate. Given suitable parameters, linear or two-dimensional arrays of allosteric subunits will then behave similar to an Ising model, exhibiting hypersharp responses to external conditions. Predictions based on this concept find good quantitative agreement in a number of experimental systems including switching of the bacterial flagellar motor, amplification of ligand signals in the Escherichia coli chemotaxis receptors, and termination of calcium sparks in cardiac muscle. A similar mechanism could potentially provide a universal mechanism of integration within living cells.
Graphical abstract Highlights ► A statistical view leads to a realistic picture of protein cooperativity. ► It allows conformational changes to propagate through a complex. ► Arrays of allosteric subunits can behave similar to an Ising model. ► Predictions based on this concept find experimental support.
Source:Journal of Molecular Biology, Volume 425, Issue 9
Author(s): Dennis Bray
A statistical view of allostery leads to a more nuanced and physically realistic picture of protein cooperativity. If the conformational state of one protein molecule in a multiprotein complex influences the probability of a particular conformation in a neighbouring protein, then changes can propagate. Given suitable parameters, linear or two-dimensional arrays of allosteric subunits will then behave similar to an Ising model, exhibiting hypersharp responses to external conditions. Predictions based on this concept find good quantitative agreement in a number of experimental systems including switching of the bacterial flagellar motor, amplification of ligand signals in the Escherichia coli chemotaxis receptors, and termination of calcium sparks in cardiac muscle. A similar mechanism could potentially provide a universal mechanism of integration within living cells.
Graphical abstract Highlights ► A statistical view leads to a realistic picture of protein cooperativity. ► It allows conformational changes to propagate through a complex. ► Arrays of allosteric subunits can behave similar to an Ising model. ► Predictions based on this concept find experimental support.
Categories: Journal Articles
Allosteric Effects in the Regulation of 26S Proteasome Activities
[Apr 2013]
Publication date: 13 May 2013
Source:Journal of Molecular Biology, Volume 425, Issue 9
Author(s): Paweł Śledź , Friedrich Förster , Wolfgang Baumeister
The 26S proteasome is the executive arm of the ubiquitin-proteasome system. This 2.5-MDa complex comprising the 20S core particle (CP) and the 19S regulatory particle (RP) is able to effectively execute its function due to a tightly regulated network of allosteric interactions. From this perspective, we summarize the current state of knowledge on these regulatory interdependencies. We classify them into the three functional layers—within the CP, within the RP, and at the CP–RP interface. In the CP, allosteric effects are thought to couple the gate opening and substrate proteolysis. Gate opening depends on events occurring in the RP—ATP hydrolysis and substrate binding. Finally, a number of processes occurring solely in the RP, like ATP hydrolysis or substrate deubiquitylation, are also proposed to be allosterically regulated. Recent advances in structural studies of 26S proteasome open up new avenues for dissecting and rationalizing the molecular basis of these regulatory networks.
Graphical abstract Highlights ► 26S proteasome executes its function in proteostasis, thanks to the allosteric regulation. ► Several distinct layers of regulation are present within the 26S proteasome. ► Cryo-electron microscopy to elucidate the mechanisms of allosteric events within the proteasome.
Source:Journal of Molecular Biology, Volume 425, Issue 9
Author(s): Paweł Śledź , Friedrich Förster , Wolfgang Baumeister
The 26S proteasome is the executive arm of the ubiquitin-proteasome system. This 2.5-MDa complex comprising the 20S core particle (CP) and the 19S regulatory particle (RP) is able to effectively execute its function due to a tightly regulated network of allosteric interactions. From this perspective, we summarize the current state of knowledge on these regulatory interdependencies. We classify them into the three functional layers—within the CP, within the RP, and at the CP–RP interface. In the CP, allosteric effects are thought to couple the gate opening and substrate proteolysis. Gate opening depends on events occurring in the RP—ATP hydrolysis and substrate binding. Finally, a number of processes occurring solely in the RP, like ATP hydrolysis or substrate deubiquitylation, are also proposed to be allosterically regulated. Recent advances in structural studies of 26S proteasome open up new avenues for dissecting and rationalizing the molecular basis of these regulatory networks.
Graphical abstract Highlights ► 26S proteasome executes its function in proteostasis, thanks to the allosteric regulation. ► Several distinct layers of regulation are present within the 26S proteasome. ► Cryo-electron microscopy to elucidate the mechanisms of allosteric events within the proteasome.
Categories: Journal Articles
Cooperativity of Allosteric Receptors
[Apr 2013]
Publication date: 13 May 2013
Source:Journal of Molecular Biology, Volume 425, Issue 9
Author(s): Stuart J. Edelstein , Nicolas Le Novère
Cooperativity of ligand binding to allosteric receptors can be quantified using the Hill coefficient (n H) to measure the sigmoidal character of the binding curve. However, for measurements of the transition between conformational states, n H values can be misleading due to ambiguity of the reference state. For cooperative ligand binding, the reference state is a hyperbolic curve for a monomer with a single binding site characterized by n H =1. Therefore, binding curves with n H >1 provide a direct measure of cooperativity. For the dependence of the conformational state on ligand concentration, curves with n H>1 are observed, but in virtually all cases, the equivalent allosteric monomer has a value of n H <1. The ratio of the two n H values defines the effective cooperativity and always corresponds to n H = N (the number of protomers in the oligomer) for concerted transitions as specified by the Monod–Wyman–Changeux model. Dose–response curves for homopentameric α7 nicotinic receptors illustrate this relationship for both wild-type and mutant forms. For functional allosteric monomers such as G-protein-coupled receptors, normalization stretches the dose–response curve along the y-axis, thereby masking the “allosteric range” and increasing the apparent cooperativity to a limit for monomers of n H =1. The concepts of equivalent monomer and allosteric range were originally proposed in 1965 by Crick and Wyman in a manuscript circulated among the proponents of allostery, but only now published for the first time in this special issue.
Graphical abstract Highlights ► The n H values for conformational transitions require a reference state. ► For conformational transitions with n H >1, for the equivalent monomer: n H <1. ► The true ratio of oligomer to monomer n H values is equal to the number of monomers. ► Normalization stretches the dose–response curve and increases the value of n H. ► The equivalent monomer concept was first proposed in 1965 by Crick and Wyman.
Source:Journal of Molecular Biology, Volume 425, Issue 9
Author(s): Stuart J. Edelstein , Nicolas Le Novère
Cooperativity of ligand binding to allosteric receptors can be quantified using the Hill coefficient (n H) to measure the sigmoidal character of the binding curve. However, for measurements of the transition between conformational states, n H values can be misleading due to ambiguity of the reference state. For cooperative ligand binding, the reference state is a hyperbolic curve for a monomer with a single binding site characterized by n H =1. Therefore, binding curves with n H >1 provide a direct measure of cooperativity. For the dependence of the conformational state on ligand concentration, curves with n H>1 are observed, but in virtually all cases, the equivalent allosteric monomer has a value of n H <1. The ratio of the two n H values defines the effective cooperativity and always corresponds to n H = N (the number of protomers in the oligomer) for concerted transitions as specified by the Monod–Wyman–Changeux model. Dose–response curves for homopentameric α7 nicotinic receptors illustrate this relationship for both wild-type and mutant forms. For functional allosteric monomers such as G-protein-coupled receptors, normalization stretches the dose–response curve along the y-axis, thereby masking the “allosteric range” and increasing the apparent cooperativity to a limit for monomers of n H =1. The concepts of equivalent monomer and allosteric range were originally proposed in 1965 by Crick and Wyman in a manuscript circulated among the proponents of allostery, but only now published for the first time in this special issue.
Graphical abstract Highlights ► The n H values for conformational transitions require a reference state. ► For conformational transitions with n H >1, for the equivalent monomer: n H <1. ► The true ratio of oligomer to monomer n H values is equal to the number of monomers. ► Normalization stretches the dose–response curve and increases the value of n H. ► The equivalent monomer concept was first proposed in 1965 by Crick and Wyman.
Categories: Journal Articles
Statistical Mechanics of Monod–Wyman–Changeux (MWC) Models
[Apr 2013]
Publication date: 13 May 2013
Source:Journal of Molecular Biology, Volume 425, Issue 9
Author(s): Sarah Marzen , Hernan G. Garcia , Rob Phillips
The 50th anniversary of the classic Monod–Wyman–Changeux (MWC) model provides an opportunity to survey the broader conceptual and quantitative implications of this quintessential biophysical model. With the use of statistical mechanics, the mathematical implementation of the MWC concept links problems that seem otherwise to have no ostensible biological connection including ligand–receptor binding, ligand-gated ion channels, chemotaxis, chromatin structure and gene regulation. Hence, a thorough mathematical analysis of the MWC model can illuminate the performance limits of a number of unrelated biological systems in one stroke. The goal of our review is twofold. First, we describe in detail the general physical principles that are used to derive the activity of MWC molecules as a function of their regulatory ligands. Second, we illustrate the power of ideas from information theory and dynamical systems for quantifying how well the output of MWC molecules tracks their sensory input, giving a sense of the “design” constraints faced by these receptors.
Graphical abstract Highlights ► The MWC concepts have broad biological reach. ► Statistical mechanics links indirect regulation to cooperativity in MWC. ► We analyzed the theoretical limitations on the performance of MWC molecules. ► A unified mathematical MWC framework links ostensibly unrelated problems.
Source:Journal of Molecular Biology, Volume 425, Issue 9
Author(s): Sarah Marzen , Hernan G. Garcia , Rob Phillips
The 50th anniversary of the classic Monod–Wyman–Changeux (MWC) model provides an opportunity to survey the broader conceptual and quantitative implications of this quintessential biophysical model. With the use of statistical mechanics, the mathematical implementation of the MWC concept links problems that seem otherwise to have no ostensible biological connection including ligand–receptor binding, ligand-gated ion channels, chemotaxis, chromatin structure and gene regulation. Hence, a thorough mathematical analysis of the MWC model can illuminate the performance limits of a number of unrelated biological systems in one stroke. The goal of our review is twofold. First, we describe in detail the general physical principles that are used to derive the activity of MWC molecules as a function of their regulatory ligands. Second, we illustrate the power of ideas from information theory and dynamical systems for quantifying how well the output of MWC molecules tracks their sensory input, giving a sense of the “design” constraints faced by these receptors.
Graphical abstract Highlights ► The MWC concepts have broad biological reach. ► Statistical mechanics links indirect regulation to cooperativity in MWC. ► We analyzed the theoretical limitations on the performance of MWC molecules. ► A unified mathematical MWC framework links ostensibly unrelated problems.
Categories: Journal Articles
The Energy and Work of a Ligand-Gated Ion Channel
[Apr 2013]
Publication date: 13 May 2013
Source:Journal of Molecular Biology, Volume 425, Issue 9
Author(s): Anthony Auerbach
Ligand-gated ion channels are allosteric membrane proteins that isomerize between C(losed) and O(pen) conformations. A difference in affinity for ligands in the two states influences the C↔O “gating” equilibrium constant. The energies associated with adult-type mouse neuromuscular nicotinic acetylcholine receptor (AChR) channel gating have been measured by using single-channel electrophysiology. Without ligands, the free energy, enthalpy and entropy of gating are ΔG 0 =+8.4, ΔH 0 =+10.9 and TΔS 0 =+2.5kcal/mol (−100mV, 23°C). Many mutations throughout the protein change ΔG 0, including natural ones that cause disease. Agonists and most mutations change approximately independently the ground-state energy difference; thus, it is possible to forecast and engineer AChR responses simply by combining perturbations. The free energy of the low↔high affinity change for the neurotransmitter at each of two functionally equivalent binding sites is ΔG B ACh =−5.1kcal/mol. ΔG B ACh is set mainly by interactions of ACh with just three binding site aromatic groups. For a series of structurally related agonists, there is a correlation between the energies of low- and high-affinity binding, which implies that gating commences with the formation of the low-affinity complex. Brief, intermediate states in binding and gating have been detected. Several proposals for the nature of the gating transition-state energy landscape and the isomerization mechanism are discussed.
Graphical abstract Highlights ► Allostery in the AChR can be studied in single molecules with electrophysiology. ► The intrinsic energy changes of the “gating” without ligands are known. ► Most mutations change the intrinsic energy, not that from the ligand. ► The effects of most mutations are additive. ► The ligand energy is from interactions with just three amino acids.
Source:Journal of Molecular Biology, Volume 425, Issue 9
Author(s): Anthony Auerbach
Ligand-gated ion channels are allosteric membrane proteins that isomerize between C(losed) and O(pen) conformations. A difference in affinity for ligands in the two states influences the C↔O “gating” equilibrium constant. The energies associated with adult-type mouse neuromuscular nicotinic acetylcholine receptor (AChR) channel gating have been measured by using single-channel electrophysiology. Without ligands, the free energy, enthalpy and entropy of gating are ΔG 0 =+8.4, ΔH 0 =+10.9 and TΔS 0 =+2.5kcal/mol (−100mV, 23°C). Many mutations throughout the protein change ΔG 0, including natural ones that cause disease. Agonists and most mutations change approximately independently the ground-state energy difference; thus, it is possible to forecast and engineer AChR responses simply by combining perturbations. The free energy of the low↔high affinity change for the neurotransmitter at each of two functionally equivalent binding sites is ΔG B ACh =−5.1kcal/mol. ΔG B ACh is set mainly by interactions of ACh with just three binding site aromatic groups. For a series of structurally related agonists, there is a correlation between the energies of low- and high-affinity binding, which implies that gating commences with the formation of the low-affinity complex. Brief, intermediate states in binding and gating have been detected. Several proposals for the nature of the gating transition-state energy landscape and the isomerization mechanism are discussed.
Graphical abstract Highlights ► Allostery in the AChR can be studied in single molecules with electrophysiology. ► The intrinsic energy changes of the “gating” without ligands are known. ► Most mutations change the intrinsic energy, not that from the ligand. ► The effects of most mutations are additive. ► The ligand energy is from interactions with just three amino acids.
Categories: Journal Articles
