Exploring the peptide 3<sub>10</sub>‐helix ⇆ α‐helix equilibrium with double label electron spin resonance

Fiori, Wayne R.; Millhauser, Glenn L.

doi:10.1002/bip.360370609

Cited by 7 publications

(7 citation statements)

References 26 publications

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“…Several such side chain interactions can be identified in the alamethicin ␣-helical starting structure (not shown), but these do not induce complete transition of ␣-helical hydrogen bonding to 3 10 helix. While recent studies with spin-labeled peptides has suggested that 3 10 -helical conformations may make a greater contribution to the structures of isolated helical polypeptides than previously thought (Fiori and Millhauser, 1995), alamethicin (and melittin) retains a largely ␣-helical conformation throughout the simulations. If the dominant polypeptide helical structure was 3 10 helix, its extent should be maximized in peptides in nonaqueous solution (like methanol) where the extra intramolecular helical hydrogen bond is favored.…”

Section: Figure 11mentioning

confidence: 85%

Hydrogen Bonding in Helical Polypeptides from Molecular Dynamics Simulations and Amide Hydrogen Exchange Analysis: Alamethicin and Melittin in Methanol

Sessions

Gibbs

Dempsey

1998

Biophysical Journal

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Molecular dynamics simulations of ion channel peptides alamethicin and melittin, solvated in methanol at 27 degrees C, were run with either regular alpha-helical starting structures (alamethicin, 1 ns; melittin 500 ps either with or without chloride counterions), or with the x-ray crystal coordinates of alamethicin as a starting structure (1 ns). The hydrogen bond patterns and stabilities were characterized by analysis of the dynamics trajectories with specified hydrogen bond angle and distance criteria, and were compared with hydrogen bond patterns and stabilities previously determined from high-resolution NMR structural analysis and amide hydrogen exchange measurements in methanol. The two alamethicin simulations rapidly converged to a persistent hydrogen bond pattern with a high level of 3(10) hydrogen bonding involving the amide NH's of residues 3, 4, 9, 15, and 18. The 3(10) hydrogen bonds stabilizing amide NH's of residues C-terminal to P2 and P14 were previously proposed to explain their high amide exchange stabilities. The absence, or low levels of 3(10) hydrogen bonds at the N-terminus or for A15 NH, respectively, in the melittin simulations, is also consistent with interpretations from amide exchange analysis. Perturbation of helical hydrogen bonding in the residues before P14 (Aib10-P14, alamethicin; T11-P14, melittin) was characterized in both peptides by variable hydrogen bond patterns that included pi and gamma hydrogen bonds. The general agreement in hydrogen bond patterns determined in the simulations and from spectroscopic analysis indicates that with suitable conditions (including solvent composition and counterions where required), local hydrogen-bonded secondary structure in helical peptides may be predicted from dynamics simulations from alpha-helical starting structures. Each peptide, particularly alamethicin, underwent some large amplitude structural fluctuations in which several hydrogen bonds were cooperatively broken. The recovery of the persistent hydrogen bonding patterns after these fluctuations demonstrates the stability of intramolecular hydrogen-bonded secondary structure in methanol (consistent with spectroscopic observations), and is promising for simulations on extended timescales to characterize the nature of the backbone fluctuations that underlie amide exchange from isolated helical polypeptides.

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Section: Figure 11mentioning

confidence: 85%

Hydrogen Bonding in Helical Polypeptides from Molecular Dynamics Simulations and Amide Hydrogen Exchange Analysis: Alamethicin and Melittin in Methanol

Sessions

Gibbs

Dempsey

1998

Biophysical Journal

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“…The experiments presented below involve detection of spin-spin interaction in mutants of T4L containing two nitroxide side chains. As discussed below, the primary mechanisms of spin-spin interaction in the cases considered here are weak through-space exchange (Fiori et al, 1993;Fiori & Millhauser, 1995), static-dipolar (Farahbakhsh et al, 1995;Rabenstein & Shin, 1995), and modulation of the dipolar interaction by molecular tumbling (Abragam, 1961), each of which leads to distance-dependent broadening of the EPR spectrum in an isotropic distribution of nitroxides. Such spectral broadening due to spin-spin interaction is readily detected by an amplitude decrease (and line width increase) in the EPR spectrum of the doubly labeled mutant with respect to the sum of the EPR spectra of the single mutants.…”

Section: Methodsmentioning

confidence: 99%

Conformation of T4 Lysozyme in Solution. Hinge-Bending Motion and the Substrate-Induced Conformational Transition Studied by Site-Directed Spin Labeling

et al. 1997

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T4 lysozyme and mutants thereof crystallize in different conformations that are related to each other by a bend about a hinge in the molecule. This observation suggests that the wild type protein may undergo a hinge-bending motion in solution to allow substrate access to an otherwise closed active site cleft [Faber, H.R., & Matthews, B.W. (1990) Nature 348, 263-266]. To test this hypothesis, either single or pairs of nitroxide side chains were introduced into the protein to monitor tertiary contact interactions and inter-residue distances, respectively, in solution. A set of constraints for these structural parameters was derived from a reference state, a covalent enzyme-substrate adduct where the enzyme is locked in the closed state. In the absence of substrate, differences in both inter-residue distances and tertiary contact interactions relative to this reference state are consistent with a hinge-bending motion that opens the active site cleft. Quantitative analysis of spin-spin interactions between nitroxide pairs reveals an 8 A relative domain movement upon substrate binding. In addition, it is demonstrated that the I3P mutation, which produces a large hinge-bending angle in the crystal, has no effect on the solution conformation. Thus, the hinge motion is not the result of the mutation but is an integral part of T4 lysozyme catalysis in solution, as suggested recently [Zhang, X.J., Wozniak, J.A., & Matthews, B.W. (1995) J. Mol. Biol. 250, 527-552]. The strategy employed here, based on site-directed spin labeling, should be generally applicable to the study of protein conformation and conformational changes in solution.

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“…The lower limit for reliable distance determination using the above methods is given by the increasing influence of exchange interaction with decreasing inter-spin distances due to partial overlap of the nitrogen π-orbitals of the two interacting nitroxides. This effect makes quantification of inter-spin distances of less than 0.8 nm difficult (48,49). In particular, a simplified inter-spin distance determination using the difference of spectral second moments…”

Section: Inter-nitroxide Distance Measurementmentioning

confidence: 99%

Methods for study of protein dynamics and protein-protein interaction in protein-ubiquitination by electron paramagnetic resonance spectroscopy

Steinhoff¹

2002

Front Biosci

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Ubiquitination is a post-translation modification whereby the C-terminal end of ubiquitin (Ub) is covalently attached to the amino group of a lysine in a target protein. Additional ubiquitin groups are added using Ub-Ub linkages to form a polyubiquitin chain. A 26S protease complex specifically binds polyubiquitinated proteins and degrades them in an ATP-dependent manner. The target lysine in the substrate protein resides in a domain that is recognized by the ubiquitination machinery in a temporally and spatially controlled manner. The accessibility and the molecular dynamics of the target domain for each protein substrate is expected to be distinctive and this article is intended to facilitate investigations in this uncharted research area of ubiquitination mediated protein turnover by means of site-directed spin labeling. Examples illustrate the methodology of electron paramagnetic resonance data acquisition and interpretation in terms of secondary and tertiary structure resolution of proteins and protein complexes. Analysis of the spin labeled side chain mobility, its solvent accessibility, the polarity of the spin label micro-environment and distances between spin labels allow to model protein domains or protein-protein interaction sites and their conformational changes with a spatial resolution at the level of the backbone fold. The structural changes accompanying protein function or protein-protein interaction can be monitored in the millisecond time range. These features make site-directed spin labeling an attractive approach for the study of protein--ubiquitin interaction and protein ubiquitination.

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Exploring the peptide 3₁₀‐helix ⇆ α‐helix equilibrium with double label electron spin resonance

Cited by 7 publications

References 26 publications

Hydrogen Bonding in Helical Polypeptides from Molecular Dynamics Simulations and Amide Hydrogen Exchange Analysis: Alamethicin and Melittin in Methanol

Hydrogen Bonding in Helical Polypeptides from Molecular Dynamics Simulations and Amide Hydrogen Exchange Analysis: Alamethicin and Melittin in Methanol

Conformation of T4 Lysozyme in Solution. Hinge-Bending Motion and the Substrate-Induced Conformational Transition Studied by Site-Directed Spin Labeling

Methods for study of protein dynamics and protein-protein interaction in protein-ubiquitination by electron paramagnetic resonance spectroscopy

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Exploring the peptide 310‐helix ⇆ α‐helix equilibrium with double label electron spin resonance

Cited by 7 publications

References 26 publications

Hydrogen Bonding in Helical Polypeptides from Molecular Dynamics Simulations and Amide Hydrogen Exchange Analysis: Alamethicin and Melittin in Methanol

Hydrogen Bonding in Helical Polypeptides from Molecular Dynamics Simulations and Amide Hydrogen Exchange Analysis: Alamethicin and Melittin in Methanol

Conformation of T4 Lysozyme in Solution. Hinge-Bending Motion and the Substrate-Induced Conformational Transition Studied by Site-Directed Spin Labeling

Methods for study of protein dynamics and protein-protein interaction in protein-ubiquitination by electron paramagnetic resonance spectroscopy

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Exploring the peptide 3₁₀‐helix ⇆ α‐helix equilibrium with double label electron spin resonance