Mapping the lifetimes of local opening events in a native state protein

Abstract

The rate constants for the processes that lead to local opening and closing of the structures around hydrogen bonds in native proteins have been determined for most of the secondary structure hydrogen bonds in the four-helix protein acyl coenzyme A binding protein. In an analysis that combines these results with the energies of activation of the opening processes and the stability of the local structures, three groups of residues in the protein structure have been identified. In one group, the structures around the hydrogen bonds have frequent openings, every 600 to 1,500 s, and long lifetimes in the open state, around 1 s. In another group of local structures, the local opening is a very rare event that takes place only every 15 to 60 h. For these the lifetime in the open state is also around 1 s. The majority of local structures have lifetimes between 2,000 and 20,000 s and relatively short lifetimes of the open state in the range between 30 and 400 ms. Mapping of these groups of amides to the tertiary structure shows that the openings of the local structures are not cooperative at native conditions, and they rarely if ever lead to global unfolding. The results suggest a mechanism of hydrogen exchange by progressive local openings.

The inverted chevron plot measured by NMR relaxation reveals a native-like unfolding intermediate in acyl-CoA binding protein

“…Clearly, the difference in structure between N and I is subtle, as gauged from the chemical shifts and other spectroscopic probes. The temperature dependence of the 1 H N chemical shifts suggests that the structural changes between N and I occur primarily in helix 3 and to some extent in helix 1, which is consistent both with the previous observation that the hydrogen bonds in helices 1 and 3 generally are less stable than those in the rest of ACBP (35) and with the 20% decrease in the near-UV CD signal of I with respect to N, which is mainly due to the two Trp residues in helix 3. Furthermore, mutations of residues in helix 3 do not substantially change the kinetics of the folding process (23), suggesting that this helix is formed late in the folding process.…”

“…Importantly, the three-state model thus reconciles the apparent discrepancy between previous observations that the global stability measured by hydrogen exchange experiments at 25°C is Ϸ5.5 kJ⅐mol Ϫ1 higher than the stability obtained from equilibrium unfolding experiments (35). The fitted value of K IN obtained in the current study corresponds to ⌬G NI ϭ 5.7 kJ⅐mol Ϫ1 at 25°C, in excellent agreement with the hydrogen exchange results.…”

“…Although the folding equilibrium is well determined by hydrogen exchange, the kinetics is less reliable. Previous estimates of folding kinetics from hydrogen exchange experiments yield much lower rates than those found both in this and other studies of ACBP folding kinetics (35). These differences are most likely due to difficulties in measuring hydrogen exchange rates in the true EX1 limit.…”

A kinetically significant intermediate in the folding of barnase

“…7 when k c ϳ k int is complicated because the general solution involves a quadratic equation with two roots (29,32). But, adequate equations can be obtained for exchange at equilibrium by using Cleland's method of net rate constants (33) and the procedure of summing transit times (ref.…”

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“…It has long been recognized that protein conformational stabilities can potentially be estimated from the rate constants of exchange of the most slowly exchanging amide hydrogens 1 . Some recent studies support this idea 2-5 , whereas other studies disagree [6][7][8] . We show here that if the effects of D 2 O and proline isomerization are considered, the conformational stabilities calculated from hydrogen exchange rate constants are in excellent agreement with those determined by traditional methods.…”