The gramicidin dimer shows both EX1 and EX2 mechanisms of H/D exchange

Abstract

We describe the use of H/D amide exchange and electrospray ionization mass spectrometry to study, in organic solvents, the pentadecapeptide gramicidin as a model for protein self association. In methanol-OD, all active H's in the peptide exchange for D within 5 min, indicating a monomer/dimer equilibrium that is shifted towards the fast-exchanging monomer. H/D exchange in n-propanol-OD, however, showed a partially protected gramicidin that slowly converts to a second species that exchanges nearly all the active hydrogens, indicating EX1 kinetics for the H/D exchange. We propose that this behavior is the result of the slower rate of unfolding in n-propanol compared with that in methanol. The rate constant for the unfolding of the dimer is the rate of disappearance of the partially protected species, and it agrees within a factor of two with a value reported in literature. The rate constant of dimer refolding can be determined from the ratio of the rate constant for unfolding and the affinity constant for the dimer, which we determined in an earlier study. The unfolding activation energy is 20 kcal mol(-1), determined by performing the exchange experiments as a function of temperature. To study gramicidin in an even more hydrophobic medium than n-propanol, we measured its H/D exchange kinetics in a phospholipids vesicle and found a different H/D amide exchange behavior. Gramicidin is an unusual peptide dimer that can exhibit both EX1 and EX2 mechanisms for its H/D exchange, depending on the solvent.

Figure 1

Mass spectral region showing (M + Na)⁺ and (M₂ + 2Na)²⁺ of gramicidin A in methanol before (a.) and after (b.) 5 min of exchange in methanol-OD. The data, acquired ~ 5 min after mixing, show nearly complete exchange of the amide hydrogens in the molecule.

Figure 2

(M₂ + 2Na)²⁺ region of gramicidin A exchanging in propanol before (a.) and after (b.) 1 h of mixing in n-propanol-OD. The data show the uptake of ~ 12 D’s out of a possible 42 in the dimer.

Figure 3

3D plot of the evolution of D-distribution as a function of time for 2 h following mixing in n-propanol-OD. Removing the native C-13 isotope distribution from the data at all the time points reveals an envelope that represents the deuterium distribution. Int is signal intensity, D is the number of deuteria taken up by the dimer, and t is the time of exchange in min.

Figure 4

Three-dimensional view of the time-dependent evolution of the D-distribution for HDX of (GA2 + 3Na)³⁺ in n-propanol-OD. The plot shows the evolution of a second-species at long times. Int is signal intensity, D is the number of deuteria taken up by the dimer, and t of exchange is time in minutes. The low intensity distributions at approximately 20 D’s at t = 0 may represent an impurity.

Figure 5

Disappearance of the partially protected species, representing unfolding of the dimer. The points are the experimental data, and the curve is the first order kinetic fit to the data.

Figure 6

Temperature dependence of the rate constant for the dimer dissociation of gramicidin in n-propanol-OD. The points are the experimental data, and the line is the linear-regression fit obtained using Microsoft Excel.