P.F.S.R.L. - Proline Isomerization



Abstract

The in vitro reactivation of E. coli Alkaline Phosphatase (AP) from the GuHCl denatured state is characterized by both fast and slow phases in activity recovery. Additionally, there exist very slow post-activation conformational changes, where the global protein lability, probed by the susceptibility of the enzyme to GuHCl denaturation, slowly decreases with time. These slow structural changes imply that the folding is initially directed by kinetic rather than thermodynamic control. This may also be evidence for folding on the "energy landscape" where there exists an ensemble of similar non-native active structures near the bottom of the thermodynamic energy "funnel". Slow events in folding have often been attributed to cis-trans isomerizations of X-Pro peptide bonds. This is a plausible explanation for AP, which contains 21 prolines per monomer. To investigate this possibility, we have performed 'triple jump' GuHCl denaturation/renaturation experiments. Our measurements of activity recovery and the evolution of lability of refolded AP as a function of denaturation time show that it is unlikely that proline isomerization is the cause of these slow events in the refolding of AP. We are investigating other possible causes of these novel slow changes, which include dimerization, domain rearrangements, metal binding, and disulfide-restricted chain rearrangements.


Introduction

Previous studies in our laboratory have shown that there exist late conformational changes in Alkaline Phosphatase (AP), refolded from the guanidine-hydrochloride (GuHCl) denatured state, on a time scale considerably longer than the recovery of enzymatic activity 1. Slow conformational changes during folding have in many cases been attributed to slow isomerizations of X-Pro peptide bonds2. We considered this to be a strong possibility for AP folding as well, since AP contains 21 prolines per monomer.
We have performed "double jump" denaturation/renaturation experiments with a third Rtriple jumpS added to determine whether proline isomerizations are the cause of the observed annealing. For different denaturation times, differences in the evolution of the GuHCl lability after activity recovery would reveal whether proline isomerization keeps the AP in a more labile form over long periods of time. We have also observed slow phases in initial activity recovery, which may also be due to proline isomerization. Differences in the recovery of activity, for different denaturation times, would indicate that proline isomerization has an effect on this process.
We also considered the possibility that other events, such as metal binding, would cause the annealing. AP requires two Zn2+ and one Mg2+ per active site for enzymatic activity and structural stability. These metals bind to monomers that are mostly folded and dimerized. We tested this possibility by analyzing the lability behavior after adding metals back to folded apo-AP.


Alkaline Phosphatase And Proline Isomerization

AP is a 94kDa homodimer, containing 2 Zn2+ and 1 Mg2+ per monomer3. It is a non-specific phosphomonoesterase that folds, dimerizes, and becomes active in the periplasm of E. coli. AP contains 21 prolines per monomer, all in the trans configuration. Certainly not all AP proline isomerizations would be expected to affect refolding or lability. The prolines that are surface exposed will likely have little affect, and the buried ones would be the most critical.
Slow folding phases have often been shown to be due to slow cis-trans isomerizations of X-Pro peptide bonds2. . Nearly all of the proline peptide bonds in native folded proteins are in the trans form. When a protein is unfolded, non-proline peptide bonds reach an equilibrium condition where trans is favored over cis by 1000:1. However, proline peptide bonds have an equilibrium of only 4:1, as the two configurations are not very different energetically4.



As a protein unfolds, the proline bonds become free to isomerize. Initially, all the prolines will remain in their native configuration, and the protein is in its "fast folding" form (upon refolding, there is no slow isomerizations that occur). However, for long denaturation times, the isomerization equilibrium will be reached, and "slow-folding" species may be produced. Refolding would then yield a slower folding phase, as the prolines must isomerize prior to the formation of the native structure.
Individual proline isomerization rates can vary widely, depending on sequence, chain length, and the presence of conformationally restrictive disulfide bonds5. Most of the protein proline isomerization results have been obtained from studies on bovine RNase A, and we will use these rates as an estimate for the isomerization rates for AP prolines. At 0 C, it has been found from double jump experiments that the time for cis-trans isomerization of the Y92-P93 bond is 900s6. Other double jump experiments at 10.5 C have shown a cis-trans isomerization time of 180s7. Adjusting to 0 C using the 20 kcal/mol activation energy yields a isomerization time constant of 709s. We have used these values as an estimate for the characteristic isomerization time between trans and cis prolines in AP at 0 C. Calculations of the number of expected cis prolines during the course of denaturation are shown below. We have assumed an equilibrium cis level of approximately 20%2.



Isomerization equation: #Cis = 4.2(1-e-t/t), t is (1) 900s or (2) 709s.


Triple Jump Experiments

The high isomerization activation energy for X-Pro peptide bonds of 20kcal/mol2. provides a useful method to determine if proline isomerization is a factor in the refolding. One can unfold Native (N) protein at very low temperatures to initially populate the unfolded (U), non-isomerized species. Over time, the prolines will isomerize to equilibrium.


Renaturation to the refolded (R) state is monitored, and annealing to the refolded/annealed (Ra) state is followed with 4.5M GuHCl lability experiments. Performing these as a function of denaturation time should reveal differences only if two conditions are satisfied:


AP Lability

AP samples that had been denatured for various times and then reactivated were allowed to refold for 1 week. 4.5M GuHCl lability curves over time show the same two lability phases, with a shift in AP population from the more to the less labile state. AP thus łanneals˛ to the less labile state.

After denaturation for various times, all samples are refolded for 2 hours and then subjected to denaturation again at high GuHCl. The subsequent activity decline is biphasic and represents 2 AP populations of different unfolding activation energies. All Refolding curves (blue) have a more labile phase of 90%, while Control has a 40% more labile phase. Unfolding and refolding AP thus transfers 50% of the AP to the more labile population. The similarity of phase populations for the Refolding samples indicates that the time of denaturation, and thus proline isomerization, appears to have little effect on the lability.



Lability Lifetimes

The fraction of more labile phase over refolding time is plotted and fit to a single exponential to determine the annealing time for each AP sample. The annealing times are very similar for all samples, as shown. Initial denaturation time, and thus proline isomerization, does not appear to influence the AP annealing process.



Conclusion

The triple-jump denaturation/renaturation studies we have shown indicate that proline isomerization is unlikely to be the cause of either the slow annealing or the slow reactivation events that are characteristic of the refolding of AP. The lability curves, annealing lifetimes, and reactivation curves are very similar despite representing AP with different amounts of incorrectly configured cis prolines at the start of reactivation. There is unlikely to be trapped cis prolines in refolding AP that produce these slow events.
In contrast, the binding of Zn2+ and Mg2+ to the AP active sites, necessary for activity and structural stability, appears to produce annealing behavior that is similar in timescale to that of AP that had been completely unfolded and refolded. Thus refolding appears to produce the folded-apo form that will anneal upon binding of metals. The exact structural changes that occur subsequent to metal binding are not yet known, but result in a decreased susceptibility to denaturation in high GuHCl.





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