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:
- Prolines have isomerized from trans to cis during denaturation
- Isomerization to cis slows down the folding/annealing.
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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