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William F Degrado - One of the best experts on this subject based on the ideXlab platform.
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new design of Helix Bundle peptide polymer conjugates
Biomacromolecules, 2008Co-Authors: William F Degrado, Ting XuAbstract:We present a new design of peptide−polymer conjugates where a polymer chain is covalently linked to the side chain of a Helix Bundle-forming peptide. The effect of conjugated polymer chains on the peptide structure was examined using a de novo designed three-Helix Bundle and a photoactive four-Helix Bundle. Upon attachment of poly(ethylene glycol) to the exterior of the coiled-coil Helix Bundle, the peptide secondary structure was stabilized and the tertiary structure, that is, the coiled-coil Helix Bundle, was retained. When a heme-binding peptide as an example is used, the new peptide−polymer conjugate architecture also preserves the built-in functionalities within the interior of the Helix Bundle. It is expected that the conjugated polymer chains act to mediate the interactions between the Helix Bundle and its external environment. Thus, this new peptide−polymer conjugate design strategy may open new avenues to macroscopically assemble the Helix Bundles and may enable them to function in nonbiological ...
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New Design of Helix Bundle Peptide–Polymer Conjugates
Biomacromolecules, 2008Co-Authors: William F Degrado, Ting XuAbstract:We present a new design of peptide−polymer conjugates where a polymer chain is covalently linked to the side chain of a Helix Bundle-forming peptide. The effect of conjugated polymer chains on the peptide structure was examined using a de novo designed three-Helix Bundle and a photoactive four-Helix Bundle. Upon attachment of poly(ethylene glycol) to the exterior of the coiled-coil Helix Bundle, the peptide secondary structure was stabilized and the tertiary structure, that is, the coiled-coil Helix Bundle, was retained. When a heme-binding peptide as an example is used, the new peptide−polymer conjugate architecture also preserves the built-in functionalities within the interior of the Helix Bundle. It is expected that the conjugated polymer chains act to mediate the interactions between the Helix Bundle and its external environment. Thus, this new peptide−polymer conjugate design strategy may open new avenues to macroscopically assemble the Helix Bundles and may enable them to function in nonbiological ...
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ultrafast folding of α3d a de novo designed three Helix Bundle protein
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Darwin O V Alonso, Valerie Daggett, Heinrich Roder, Kosuke Maki, Chengyen Huang, Steven J Lahr, William F DegradoAbstract:Here, we describe the folding/unfolding kinetics of α3D, a small designed three-Helix Bundle. Both IR temperature jump and ultrafast fluorescence mixing methods reveal a single-exponential process consistent with a minimal folding time of 3.2 ± 1.2 μs (at ≈50°C), indicating that a protein can fold on the 1- to 5-μs time scale. Furthermore, the single-exponential nature of the relaxation indicates that the prefactor for transition state (TS)-folding models is probably ≥1 (μs)–1 for a protein of this size and topology. Molecular dynamics simulations and IR spectroscopy provide a molecular rationale for the rapid, single-exponential folding of this protein. α3D shows a significant bias toward local helical structure in the thermally denatured state. The molecular dynamics-simulated TS ensemble is highly heterogeneous and dynamic, allowing access to the TS via multiple pathways.
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solution structure and dynamics of a de novo designed three Helix Bundle protein
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Scott T R Walsh, James W. Bryson, Hong Cheng, Heinrich Roder, William F DegradoAbstract:Although de novo protein design is an important endeavor with implications for understanding protein folding, until now, structures have been determined for only a few 25- to 30-residue designed miniproteins. Here, the NMR solution structure of a complex 73-residue three-Helix Bundle protein, α3D, is reported. The structure of α3D was not based on any natural protein, and yet it shows thermodynamic and spectroscopic properties typical of native proteins. A variety of features contribute to its unique structure, including electrostatics, the packing of a diverse set of hydrophobic side chains, and a loop that incorporates common capping motifs. Thus, it is now possible to design a complex protein with a well defined and predictable three-dimensional structure.
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From coiled coils to small globular proteins: design of a native-like three-Helix Bundle.
Protein Science, 1998Co-Authors: James W. Bryson, John R. Desjarlais, Tracy M. Handel, William F DegradoAbstract:A monomolecular native-like three-Helix Bundle has been designed in an iterative process, beginning with a peptide that noncooperatively assembled into an antiparallel three-Helix Bundle. Three versions of the protein were designed in which specific interactions were incrementally added. The hydrodynamic and spectroscopic properties of the proteins were examined by size exclusion chromatography, sedimentation equilibrium, fluorescence spectroscopy, and NMR. The thermodynamics of folding were evaluated by monitoring the thermal and guanidine-induced unfolding transitions using far UV circular dichroism spectroscopy. The attainment of a unique, native-like state was achieved through the introduction of: (1) Helix capping interactions; (2) electrostatic interactions between partially exposed charged residues; (3) a diverse collection of apolar side chains within the hydrophobic core.
Jonas S. Johansson - One of the best experts on this subject based on the ideXlab platform.
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The Effect of Alpha-Helix Linker Length on Volatile General Anesthetic Binding to the Four-Alpha-Helix Bundle (Aα2-L38M)2
Biophysical Journal, 2020Co-Authors: Lucia M. Morstadt, Jonas S. JohanssonAbstract:In order to investigate the effects of the length of the linker connecting the alpha-helices in the di-alpha-helical protein Aα2-L38M on the overall structure and on the affinity of anesthetic binding to four-alpha-Helix Bundle (Aα2-L38M)2, we designed constructs containing six and four glycine residue linkers instead of the original eight, respectively. We used site-directed mutagenesis with primers designed to remove two and four glycine residues, respectively, out of eight residues of the original glycine linker present in the four-alpha-Helix Bundle (Aα2-L38M)2. Variant proteins were expressed in bacteria and purified to homogeneity using reverse-phase HPLC. Protein identities were verified using mass spectrometry. Our initial studies reveal that the variant four-alpha-Helix Bundle with a four glycine linker binds halothane with a dissociation constant of 1.00 ± 0.05 mM, as assessed using fluorescence spectroscopy. This represents a 10-fold decreased affinity for the volatile general anesthetic compared to the original (Aα2-L38M)2 design, which featured an eight glycine linker. The results indicate that the length of the glycine linker, and the resulting dynamic behavior of the four-alpha-Helix Bundle protein, dramatically influence volatile general anesthetic binding affinity.
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design and biophysical characterization of a monomeric four alpha Helix Bundle protein aα4 with affinity for the volatile anesthetic halothane
Biochimica et Biophysica Acta, 2012Co-Authors: Lucia M. Morstadt, Qing Cheng Meng, Jonas S. JohanssonAbstract:Abstract A monomeric four-α-Helix Bundle protein Aα 4 was designed as a step towards investigating the interaction of volatile general anesthetics with their putative membrane protein targets. The alpha helices, connected by glycine loops, have the sequence A, B, B′, A′. The DNA sequence was designed to make the helices with the same amino acid sequences (Helix A and A′, B and B′, respectively) as different as possible, while using codons which are favorable for expression in E. coli . The protein was bacterially expressed and purified to homogeneity using reversed-phase HPLC. Protein identity was verified using MALDI–TOF mass spectrometry. Far-UV circular dichroism spectroscopy confirmed the predominantly alpha-helical nature of the protein Aα 4 . Guanidinium chloride induced denaturation showed that the monomeric four-α-Helix Bundle protein Aα 4 is considerably more stable compared to the dimeric di-α-helical protein (Aα 2 -L38M) 2 . The sigmoidal character of the unfolding reaction is conserved while the sharpness of the transition is increased 1.8-fold. The monomeric four-α-Helix Bundle protein Aα 4 bound halothane with a dissociation constant (K d ) of 0.93 ± 0.02 mM, as shown by both tryptophan fluorescence quenching and isothermal titration calorimetry. This monomeric four-α-Helix Bundle protein can now be used as a scaffold to incorporate natural central nervous system membrane protein sequences in order to examine general anesthetic interactions with putative targets in detail.
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Design and Biophysical Characterization of a Single Chain Four-alpha-Helix Bundle Protein Which Binds Volatile General Anesthetics
Biophysical Journal, 2011Co-Authors: Lucia M. Morstadt, Qing Cheng Meng, Jonas S. JohanssonAbstract:In order to investigate the interaction of volatile general anesthetics with their putative membrane protein targets, we designed a four-alpha-Helix Bundle protein in a single protein chain. The four alpha helices, connected by three 8 glycine loops, had the sequence A, B, B’, A’. The DNA sequence was designed with the goal of making helices with the same amino acid sequence (Helix A and A’, B and B’, respectively) as different as possible in their DNA sequence, while using codons which are favorable for expression in E.coli. Restriction enzyme sites were added on both ends. The synthesized DNA sequence was cloned into an expression vector. The protein was bacterially expressed and purified to homogeneity using reverse-phase HPLC. Protein identity was verified using MALDI-TOF mass spectroscopy. Near UV circular dichroism spectroscopy confirmed the strongly alpha helical nature of the protein. Guanidinuium chloride denaturation showed that the single chain four-alpha-Helix Bundle protein is twice as stable as the dimeric di-helical protein. The sigmoidal character of the unfolding reaction was conserved, the sharpness of the transition increased. Our single chain four-alpha-Helix Bundle protein bound halothane with a dissociation constant of 1.2 mM, as shown by tryptophan fluorescence quenching. This single chain four-alpha-Helix Bundle protein can now be used as a scaffold to incorporate natural membrane protein sequences to examine general anesthetic interactions in detail.
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Binding of the General Anesthetics Chloroform and 2,2,2-Trichloroethanol to the Hydrophobic Core of a Four–α-Helix Bundle Protein¶
Photochemistry and Photobiology, 2007Co-Authors: Jonas S. Johansson, Ken Solt, Konda S. ReddyAbstract:Abstract The structural features of general anesthetic binding sites on proteins are being examined using a defined model system consisting of a four–α-Helix Bundle scaffold with a hydrophobic core. Previous work suggested that halothane binding to the four–α-Helix Bundle was improved by (1) introducing a cavity into the hydrophobic core and (2) substituting a methionine side-chain in place of an α-helical heptad e position leucine. In this study, the ability of the general anesthetics chloroform and 2,2,2-trichloroethanol to bind to the hydrophobic core of the four–α-Helix Bundle (Aα2-L38M)2 is explored. The halogenated alkane chloroform binds with a dissociation constant (Kd) = 1.4 ± 0.2 mM, whereas 2,2,2-trichloroethanol binds with a Kd = 19.5 ± 1.2 mM. The affinity of both general anesthetics for the hydrophobic core of the four–α-Helix Bundle approximates their whole animal effective concentration in 50% of test subjects' (EC50) values, as shown previously for halothane. Tryptophan phosphorescence de...
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kinetics of anesthetic induced conformational transitions in a four alpha Helix Bundle protein
Biochemistry, 2006Co-Authors: Ken Solt, Jonas S. Johansson, Douglas E RainesAbstract:Inhaled general anesthetics alter the function of numerous proteins (1-5). Although the receptor site(s) responsible for producing anesthesia are not known with certainty, members of an anesthetic-sensitive superfamily of homologous Cys-loop ligand-gated ion channels (LGICs) are believed to be among the most important targets (6-8). Members of this superfamily include the nicotinic acetylcholine, serotonin type 3, γ-aminobutyric acid type A (GABAA), and glycine receptors (9, 10). Electrophysiological studies using receptor chimeras and site-directed mutagenesis techniques suggest that inhaled anesthetics bind to LGICs within discrete protein cavities that are lined by portions of four α-helical transmembrane domains (M1-M4), producing conformational states with increased sensitivity to agonist (11-14). In the case of the GABAA receptor, inhaled anesthetics can also directly activate the ion channel even in the absence of agonist (15, 16). Both of these actions may occur if general anesthetics stabilize receptors in an open channel state. However because Cys-loop LGICs are complex molecules that cannot be expressed in significant quantities and at high purity, there remain considerable obstacles to applying powerful biophysical and biochemical approaches to gain insight into how inhaled anesthetics act on these targets. To overcome these obstacles, small, water-soluble four-α-Helix Bundle proteins have been designed, produced, and used as well-defined model systems to explore how inhaled anesthetics bind to proteins (17-21). Each four-α-Helix Bundle protein is a dimer of two 27-residue α-helices joined by an eight-residue glycine linker (figure 1). It contains a hydrophobic cavity that is lined by the four α-helices and binds inhaled anesthetics. Recently, it has been shown that such binding can alter the protein's conformational state as evidenced by changes in its fluorescence, circular dichroism and nuclear magnetic resonance spectra following equilibration with anesthetic (22). Such observations suggest that four-α-Helix Bundle proteins can serve not only as models to explore how anesthetics bind, but also how such binding leads to changes in protein structure and function. Figure 1 (A) Modeled structure of the expressed four-α-Helix Bundle (Aα2-L1M/L38M)2. The cylinders represent the two 27-residue amphiphilic α-helical portions of each 62-residue di-α-helical peptide, joined by an eight-residue glycine ... The present study was undertaken to examine the kinetics with which inhaled anesthetics interact with the four-α-Helix Bundle protein (Aα2-L1M/L38M)2. Stopped-flow fluorescence spectroscopic techniques having millisecond time resolution were used to monitor anesthetic-protein interactions. This was possible because each peptide in (Aα2-L1M/L38M)2 contains a tryptophan residue at position 15 (W15) whose fluorescence is reduced upon equilibration with clinically relevant concentrations of anesthetic (22, 23). We focused our studies on two inhaled anesthetics, halothane and sevoflurane. Halothane directly and efficiently quenches tryptophan fluorescence (24) whereas sevoflurane does not (22).
David R Critchley - One of the best experts on this subject based on the ideXlab platform.
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activation of a vinculin binding site in the talin rod involves rearrangement of a five Helix Bundle
The EMBO Journal, 2004Co-Authors: E Papagrigoriou, Alexandre R Gingras, I L Barsukov, Neil Bate, Ian J Fillingham, Bipin Patel, Ronald Frank, Wolfgang H Ziegler, Gordon C K Roberts, David R CritchleyAbstract:The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. We have determined the crystal structures of two domains from the talin rod spanning residues 482–789. Talin 482–655, which contains a vinculin-binding site (VBS), folds into a five-Helix Bundle whereas talin 656–789 is a four-Helix Bundle. We show that the VBS is composed of a hydrophobic surface spanning five turns of Helix 4. All the key side chains from the VBS are buried and contribute to the hydrophobic core of the talin 482–655 fold. We demonstrate that the talin 482–655 five-Helix Bundle represents an inactive conformation, and mutations that disrupt the hydrophobic core or deletion of Helix 5 are required to induce an active conformation in which the VBS is exposed. We also report the crystal structure of the N-terminal vinculin head domain in complex with an activated form of talin. Activation of the VBS in talin and the recruitment of vinculin may support the maturation of small integrin/talin complexes into more stable adhesions.
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Activation of a vinculin‐binding site in the talin rod involves rearrangement of a five‐Helix Bundle
The EMBO Journal, 2004Co-Authors: E Papagrigoriou, Alexandre R Gingras, I L Barsukov, Neil Bate, Ian J Fillingham, Bipin Patel, Ronald Frank, Wolfgang H Ziegler, Gordon C K Roberts, David R CritchleyAbstract:The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. We have determined the crystal structures of two domains from the talin rod spanning residues 482–789. Talin 482–655, which contains a vinculin-binding site (VBS), folds into a five-Helix Bundle whereas talin 656–789 is a four-Helix Bundle. We show that the VBS is composed of a hydrophobic surface spanning five turns of Helix 4. All the key side chains from the VBS are buried and contribute to the hydrophobic core of the talin 482–655 fold. We demonstrate that the talin 482–655 five-Helix Bundle represents an inactive conformation, and mutations that disrupt the hydrophobic core or deletion of Helix 5 are required to induce an active conformation in which the VBS is exposed. We also report the crystal structure of the N-terminal vinculin head domain in complex with an activated form of talin. Activation of the VBS in talin and the recruitment of vinculin may support the maturation of small integrin/talin complexes into more stable adhesions.
Ting Xu - One of the best experts on this subject based on the ideXlab platform.
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amphiphilic peptide polymer conjugates based on the coiled coil Helix Bundle
Biomacromolecules, 2010Co-Authors: Yuja Huang, Andrew D Presley, Joseph J Chang, Ting XuAbstract:Amphiphilic peptide−polymer conjugates can lead to hierarchically structured, biomolecular materials. Because the peptide structure determines the size, shape, and intermolecular interactions of these building blocks, systematic understanding of how the peptide structure and functionality are affected upon implementing hydrophobicity is required to direct their assemblies in solution and in the solid state. However, depending on the peptide sequence and native structure, previous studies have shown that the hydrophobic moieties affect peptide structures differently. Here, we present a solution study of amphiphilic peptide−polymer conjugates, where a hydrophobic polymer, polystyrene, is covalently linked to the N-terminus of a coiled-coil Helix Bundle-forming peptide. The effect of conjugated hydrophobic polymers on the peptide secondary and tertiary structures was examined using two types of model, coiled-coil Helix Bundles. In particular, the integrity of the binding pocket within the Helix Bundle upon h...
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new design of Helix Bundle peptide polymer conjugates
Biomacromolecules, 2008Co-Authors: William F Degrado, Ting XuAbstract:We present a new design of peptide−polymer conjugates where a polymer chain is covalently linked to the side chain of a Helix Bundle-forming peptide. The effect of conjugated polymer chains on the peptide structure was examined using a de novo designed three-Helix Bundle and a photoactive four-Helix Bundle. Upon attachment of poly(ethylene glycol) to the exterior of the coiled-coil Helix Bundle, the peptide secondary structure was stabilized and the tertiary structure, that is, the coiled-coil Helix Bundle, was retained. When a heme-binding peptide as an example is used, the new peptide−polymer conjugate architecture also preserves the built-in functionalities within the interior of the Helix Bundle. It is expected that the conjugated polymer chains act to mediate the interactions between the Helix Bundle and its external environment. Thus, this new peptide−polymer conjugate design strategy may open new avenues to macroscopically assemble the Helix Bundles and may enable them to function in nonbiological ...
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New Design of Helix Bundle Peptide–Polymer Conjugates
Biomacromolecules, 2008Co-Authors: William F Degrado, Ting XuAbstract:We present a new design of peptide−polymer conjugates where a polymer chain is covalently linked to the side chain of a Helix Bundle-forming peptide. The effect of conjugated polymer chains on the peptide structure was examined using a de novo designed three-Helix Bundle and a photoactive four-Helix Bundle. Upon attachment of poly(ethylene glycol) to the exterior of the coiled-coil Helix Bundle, the peptide secondary structure was stabilized and the tertiary structure, that is, the coiled-coil Helix Bundle, was retained. When a heme-binding peptide as an example is used, the new peptide−polymer conjugate architecture also preserves the built-in functionalities within the interior of the Helix Bundle. It is expected that the conjugated polymer chains act to mediate the interactions between the Helix Bundle and its external environment. Thus, this new peptide−polymer conjugate design strategy may open new avenues to macroscopically assemble the Helix Bundles and may enable them to function in nonbiological ...
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Structural studies of amphiphilic 4-Helix Bundle peptides incorporating designed extended chromophores for nonlinear optical biomolecular materials.
Nano Letters, 2006Co-Authors: Joseph Strzalka, Ting Xu, Andrey Tronin, Sophia P. Wu, Ivan R. Miloradovic, Ivan Kuzmenko, Michael J. Therien, J. Kent BlasieAbstract:Extended conjugated chromophores containing (porphinato)zinc components that exhibit large optical polarizabilities and hyperpolarizabiliites are incorporated into amphiphilic 4-Helix Bundle peptides via specific axial histidyl ligation of the metal. The Bundle's designed amphiphilicity enables vectorial orientation of the chromophore/peptide complex in macroscopic monolayer ensembles. The 4-Helix Bundle structure is maintained upon incorporation of two different chromophores at stoichiometries of 1-2 per Bundle. The axial ligation site appears to effectively control the position of the chromophore along the length of the Bundle.
E Papagrigoriou - One of the best experts on this subject based on the ideXlab platform.
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activation of a vinculin binding site in the talin rod involves rearrangement of a five Helix Bundle
The EMBO Journal, 2004Co-Authors: E Papagrigoriou, Alexandre R Gingras, I L Barsukov, Neil Bate, Ian J Fillingham, Bipin Patel, Ronald Frank, Wolfgang H Ziegler, Gordon C K Roberts, David R CritchleyAbstract:The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. We have determined the crystal structures of two domains from the talin rod spanning residues 482–789. Talin 482–655, which contains a vinculin-binding site (VBS), folds into a five-Helix Bundle whereas talin 656–789 is a four-Helix Bundle. We show that the VBS is composed of a hydrophobic surface spanning five turns of Helix 4. All the key side chains from the VBS are buried and contribute to the hydrophobic core of the talin 482–655 fold. We demonstrate that the talin 482–655 five-Helix Bundle represents an inactive conformation, and mutations that disrupt the hydrophobic core or deletion of Helix 5 are required to induce an active conformation in which the VBS is exposed. We also report the crystal structure of the N-terminal vinculin head domain in complex with an activated form of talin. Activation of the VBS in talin and the recruitment of vinculin may support the maturation of small integrin/talin complexes into more stable adhesions.
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Activation of a vinculin‐binding site in the talin rod involves rearrangement of a five‐Helix Bundle
The EMBO Journal, 2004Co-Authors: E Papagrigoriou, Alexandre R Gingras, I L Barsukov, Neil Bate, Ian J Fillingham, Bipin Patel, Ronald Frank, Wolfgang H Ziegler, Gordon C K Roberts, David R CritchleyAbstract:The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. We have determined the crystal structures of two domains from the talin rod spanning residues 482–789. Talin 482–655, which contains a vinculin-binding site (VBS), folds into a five-Helix Bundle whereas talin 656–789 is a four-Helix Bundle. We show that the VBS is composed of a hydrophobic surface spanning five turns of Helix 4. All the key side chains from the VBS are buried and contribute to the hydrophobic core of the talin 482–655 fold. We demonstrate that the talin 482–655 five-Helix Bundle represents an inactive conformation, and mutations that disrupt the hydrophobic core or deletion of Helix 5 are required to induce an active conformation in which the VBS is exposed. We also report the crystal structure of the N-terminal vinculin head domain in complex with an activated form of talin. Activation of the VBS in talin and the recruitment of vinculin may support the maturation of small integrin/talin complexes into more stable adhesions.