Conclusions
-
Our intial hypothesis about the relationship between tryptophan
flexibility and RTP lifetime is qualitatively supported by the observed
RTP lifetimes of the glutamine mutants of AP [see Figure 1].
-
The biphasic RTP decays during exchange indicate that we
are dealing with two distinct, at least from a phosphorescence perspective,
populations of proteins. This postulate is also consistent with the behavior
exhibited by the relative percentage of long and short lifetimes of the
average RTP decay. An obvious candidate for H/D exchange giving rise to
these populations is the enamine hydrogen of tryptophan 109.
-
The lack of pD dependence for the exchange rates [see Figure
4] is an indication that the exchange kinetics are of the EX1 type and
therefore the rate-limiting step in the exchange is the opening of the
protein to expose the exchanging residue to the solvent. EX1 kinetics are
typically observed for only deeply buried residues. This observation is
consistent with the theory that we are monitoring the exchange at the tryptophan
109 enamine hydrogen.
-
Experiments performed with buffers containing varying concentrations
of D2O and H2O
are also consistent with the theory that it is the exchange of a single
hydrogen which is causing the change in the phosphorescence lifetime [see
Figure 5].
-
The calculated activation energies for this exchange process
for WT and Q320G are quite similar [see Figure 6] which suggests that the
slowest process in the exchange for both proteins is the same. A likely
candidate for this process is the opening of the protein exposure of the
tryptophan to solvent.
-
The difference in the exchange rates between WT and Q320G
would then argue that the specific exchange process for that hydrogen is
affected by hydrogen bonding. A similar effect of hydrogen bonding was
seen by Zhang et al in rabbit muscle aldolase and by Milne et al in cytochrome
c.
-
The large difference in both the activation energy and the
exchange rates for the hydrogen exchange between WT and Q320L is somewhat
surprising. Conclusions are difficult to make without having crystal structures
for the enzyme, but it is possible that the Q to L mutation severely disrupts
the packing of the hydrophobic core thereby destabilizing it and making
the opening of the protein for exchange occur more readily.
-
No difference is seen in calorimetric melts of the three
proteins [see below], so the global stability of all mutants is not strongly
affected by the mutations. The exchange data is thus providing us a more
localized probe of flexibility or rigidity.
| Protein |
DH (kcal/mol) |
DS (kcal/K mol) |
Tm (K) |
| WT |
419.6 |
1.139 |
95.3 |
| Q320G |
418.7 |
1.130 |
93.3 |
| Q320L |
419.7 |
1.145 |
93.3 |
References