GLOBAL TOPOLOGY AND STABILITY AND LOCAL-STRUCTURE AND DYNAMICS IN A SYNTHETIC SPIN-LABELED 4-HELIX BUNDLE PROTEIN

A maleimide nitroxide spin-label (MAL-6) linked to a cysteine in the h ydrophobic core and a coproporphyrin I (CP) appended on the N-terminus of a synthetic helix-loop-helix peptide ([alpha(2)]) have been used t o examine the designed self-association of a four-helix bundle ([alpha (2)](2)), focusing on the bundle topology and stability and the rotati onal dynamics of the spin-label. Gel-permeation chromatography demonst rated that the [alpha(2)] peptide and the peptide modified with a spin -label ([MAL-6-alpha(2)]), a coproporphyrin ([CP-alpha(2)]) and a copr oporphyrin plus a spin-label ([CP-MAL-6-alpha(2)]) self-associate into four helix bundles in solution as designed. Circular dichroism (CD) s pectra prove that all these peptides are highly or-helical, confirmed for [alpha(2)](2) by Fourier transform infrared (FTIR) spectroscopic a nalysis. Electron spin resonance (ESR) spectra of the two attached mal eimide spin-labels in [MAL-6-alpha(2)](2) shows their effective rotati onal correlation time (tau(c)) 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 aq ueous-phase paramagnetic ions, Ni(LI), demonstrating all of the spin-l abels are buried within the hydrophobic core. The lack of spin-spin in teraction between the buried, immobilized spin-labels indicates they a re remote (>15 Angstrom) from each other, indicating an antiparallel t opology of the monomers in [MAL-6-alpha(2)](2). The parent [alpha(2)]( 2) and the modified [MAL-6-alpha(2)](2) and [CP-alpha(2)](2) peptides are highly stable (Delta G(H2O) approximate to 25 kcal/mol) as investi gated by guanidine hydrochloride denaturation curves monitored by ESR and CD spectroscopies. Guanidine hydrochloride denaturation leads to a shorter correlation time of the spin-label, tau(c) < 1 ns, approachin g that of an unrestricted spin-label in solution. In contrast, trifluo roethanol caused dissociation of [MAL-6-alpha(2)](2) to yield two [MAL -6-alpha(2)] monomers with retention of secondary structure and change d the tau(c) to 2.5 +/- 0.5 ns, indicating that a significant degree o f motional restriction is imposed on the spin-label by the secondary s tructure. The coproporphyrin probes covalently attached to the N-termi ni of [Cp-alpha(2)](2) and [CP-MAL-6-alpha(2)](2) provided evidence th at the helical monomers of both were in a parallel orientation, in con trast to the antiparallel orientation determined for [MAL-6-alpha(2)]( 2). Consequently, the ESR spectra of [MAL-6-alpha(2)](2) and [CP-MAL-6 -alpha(2)](2) reveal major structural differences in the local vicinit y of the spin-labels due to the topological difference between these t wo bundles. The ESR spectra of [CP-MAL-6-alpha(2)](2) contains two dis tinct nitroxide populations, indicating that one spin-label remains bu ried in the hydrophobic core and the other is excluded to solvent in t his parallel topology. Alleviation of the steric interactions causing one spin-label in [CP-MAL-6-alpha(2)](2) to be solvent-exposed by addi tion of [CP-alpha(2)](2) results in formation of the heterodimeric [CP -alpha(2)]/[CP-MAL-6-alpha(2)], as evidenced by insertion of all the s pin-labels into hydrophobic cores. The changes in global topology and local structure as evidenced by this pair of spectral probes have rela tively minor effects on the course of guanidine denaturation of these bundles.