Ion Mobility-Mass Spectrometry Reveals the Energetics of Intermediates that Guide Polyproline Folding

Abstract

Proline favors trans-configured peptide bonds in native proteins. Although cis/trans configurations vary for non-native and unstructured states, solvent also influences these preferences. Water induces the all-cis right-handed polyproline-I (PPI) helix of polyproline to fold into the all-trans left-handed polyproline-II (PPII) helix. Our recent work has shown that this occurs via a sequential mechanism involving six resolved intermediates. Here, we use ion mobility-mass spectrometry to make the first detailed thermodynamic measurements of the folding intermediates, which inform us about how and why this transition occurs. It appears that early intermediates are energetically favorable due to hydration of the peptide backbone, while late intermediates are enthalpically unfavorable. However, folding continues, as the entropy of the system increases upon successive formation of each new structure. When PPII is immersed in 1-propanol, the PPII→PPI transition occurs, but this reaction occurs through a very different mechanism. Early on, the PPII population splits onto multiple pathways that eventually converge through a late intermediate that continues on to the folded PPI helix. Nearly every step is endothermic. Folding results from a stepwise increase in the disorder of the system, allowing a wide-scale search for a critical late intermediate. Overall, the data presented here allow us to establish the first experimentally-determined energy surface for biopolymer folding as a function of solution environment.

“Wet” Versus “Dry” Folding of Polyproline

“…Recently, we examined the kinetics and thermodynamics of structural transitions associated with a thirteen-residue polyproline (Pro13) interconverting between the PPI and PPII helices in different solution environments [26,27]. When PPI PrOH is immersed in water, the peptide undergoes a spontaneous, stepwise folding transition through six intermediates as it folds into the PPII aq form [26].…”

“…Alternatively, when the hydrated PPII aq helix is immersed in 1-propanol, multiple cis/trans -configured intermediates are formed in parallel. These different structures subsequently undergo folding transitions and converge through a series of intermediates en route to forming the PPI PrOH helix [27]. As the observed structural transitions are a direct result of the dehydration process associated with the displacement of water by 1-propanol, the PPII aq →PPI PrOH transition is referred to as a “ dry ” solution-folding.…”

“…The previously published kinetics data, used to determine each of these solution-phase folding mechanisms, are summarized in Figure 1. As explained in the figure, the kinetics fits are extremely sensitive to the employed model, allowing for delineation of detailed folding pathways [26,27]. …”

Microsecond and nanosecond polyproline II helix formation in aqueous nanodrops measured by mass spectrometry

“…20-22 Formation kinetics of PPII helices in more native-like buffered aqueous solutions, however, have not been measured. Recently, double-barrel nano-electrospray ionization (nanoESI) emitters, also known as theta-glass emitters, have been used to rapidly mix solutions during nanoESI to investigate numerous room-temperature reactions, 23-27 including monitoring protein folding reactions that occur in microseconds.…”

“…The significant difference in folding time constants obtained for these two peptides shows that the formation time of PPII helices from highly unfolded structures depends on the amino-acid sequence, consistent with results reported for the transition from PPI to PPII helices. 20-22 …”