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Global Topology & Stability and Local Structure & Dynamics in a Synthetic Spin-Labeled Four-Helix Bundle Protein

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Johnson Research Foundation, Department of Biochemistry and Biophysics, and Department of Anesthesia, University of Pennsylvania, Philadelphia, Pennsylvania 19104, and Departments of Chemistry, Biological Sciences, and Biophysical Sciences and Center for Structural Biology, State University of New York, Buffalo, New York 14260
Cite this: Biochemistry 1997, 36, 10, 2798–2806
Publication Date (Web):March 11, 1997
https://doi.org/10.1021/bi9618225
Copyright © 1997 American Chemical Society

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    Abstract

    A maleimide nitroxide spin-label (MAL-6) linked to a cysteine in the hydrophobic core and a coproporphyrin I (CP) appended on the N-terminus of a synthetic helix−loop−helix peptide ([α2]) have been used to examine the designed self-association of a four-helix bundle ([α2]2), focusing on the bundle topology and stability and the rotational dynamics of the spin-label. Gel-permeation chromatography demonstrated that the [α2] peptide and the peptide modified with a spin-label ([MAL-6-α2]), a coproporphyrin ([CP-α2]) and a coproporphyrin plus a spin-label ([CP-MAL-6-α2]) self-associate into four helix bundles in solution as designed. Circular dichroism (CD) spectra prove that all these peptides are highly α-helical, confirmed for [α2]2 by Fourier transform infrared (FTIR) spectroscopic analysis. Electron spin resonance (ESR) spectra of the two attached maleimide spin-labels in [MAL-6-α2]2 shows their effective rotational correlation time (τc) is 7.3 ± 0.5 ns, consistent with that expected for the tumbling of the four helix bundle itself, indicating the labels are immobilized. The ESR spectra were also unaltered by aqueous-phase paramagnetic ions, Ni(II), demonstrating all of the spin-labels are buried within the hydrophobic core. The lack of spin−spin interaction between the buried, immobilized spin-labels indicates they are remote (>15 Å) from each other, indicating an antiparallel topology of the monomers in [MAL-6-α2]2. The parent [α2]2 and the modified [MAL-6-α2]2 and [CP-α2]2 peptides are highly stable (ΔGH2O ≈ 25 kcal/mol) as investigated by guanidine hydrochloride denaturation curves monitored by ESR and CD spectroscopies. Guanidine hydrochloride denaturation leads to a shorter correlation time of the spin-label, τc < 1 ns, approaching that of an unrestricted spin-label in solution. In contrast, trifluoroethanol caused dissociation of [MAL-6-α2]2 to yield two [MAL-6-α2] monomers with retention of secondary structure and changed the τc to 2.5 ± 0.5 ns, indicating that a significant degree of motional restriction is imposed on the spin-label by the secondary structure. The coproporphyrin probes covalently attached to the N-termini of [CP-α2]2 and [CP-MAL-6-α2]2 provided evidence that the helical monomers of both were in a parallel orientation, in contrast to the antiparallel orientation determined for [MAL-6-α2]2. Consequently, the ESR spectra of [MAL-6-α2]2 and [CP-MAL-6-α2]2 reveal major structural differences in the local vicinity of the spin-labels due to the topological difference between these two bundles. The ESR spectra of [CP-MAL-6-α2]2 contains two distinct nitroxide populations, indicating that one spin-label remains buried in the hydrophobic core and the other is excluded to solvent in this parallel topology. Alleviation of the steric interactions causing one spin-label in [CP-MAL-6-α2]2 to be solvent-exposed by addition of [CP-α2]2 results in formation of the heterodimeric [CP-α2]/[CP-MAL-6-α2], as evidenced by insertion of all the spin-labels into hydrophobic cores. The changes in global topology and local structure as evidenced by this pair of spectral probes have relatively minor effects on the course of guanidine denaturation of these bundles.

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    This work was supported by the National Institutes of Health (Grants GM27309 and GM41048 to P.L.D. and GM 35940 to A.J.W.). B.R.G. and J.J.S. gratefully acknowledge receipt of postdoctoral fellowships from the NIH (Grants GM17816 and GM18121, respectively). J.S.J. was supported in part by a Foundation for Anesthesia Education and Research Young Investigator Award. F.R. was supported by a postdoctoral fellowship from the European Molecular Biology Organization. The infrared spectrometer is supported by the NIH (GM 48130).

     Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania.

    §

     Department of Anesthesia, University of Pennsylvania.

     State University of New York.

    *

    In papers with more than one author, the asterisk indicates the name of the author to whom inquiries about the paper should be addressed.

     Abstract published in Advance ACS Abstracts, February 15, 1997.

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