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

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 (ΔG H 2 O ≈ 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