Protein dynamics viewed by hydrogen exchange

Abstract

To examine the relationship between protein structural dynamics and measurable hydrogen exchange (HX) data, the detailed exchange behavior of most of the backbone amide hydrogens of Staphylococcal nuclease was compared with that of their neighbors, with their structural environment, and with other information. Results show that H-bonded hydrogens are protected from exchange, with HX rate effectively zero, even when they are directly adjacent to solvent. The transition to exchange competence requires a dynamic structural excursion that removes H-bond protection and allows exposure to solvent HX catalyst. The detailed data often make clear the nature of the dynamic excursion required. These range from whole molecule unfolding, through smaller cooperative unfolding reactions of secondary structural elements, and down to local fluctuations that involve as little as a single peptide group or side chain or water molecule. The particular motion that dominates the exchange of any hydrogen is the one that allows the fastest HX rate. The motion and the rate it produces are determined by surrounding structure and not by nearness to solvent or the strength of the protecting H-bond itself or its acceptor type (main chain, side chain, structurally bound water). Many of these motions occur over time scales that are appropriate for biochemical function.

Figure 1

H-bonding inventory showing measured log protection factor versus H-bond acceptor. Symbols indicate amide hydrogens known to exchange by way of a large unfolding reaction (green), or a small local fluctuation (red), or whether this information is not available (black). Amides that H-bond to solvent are separated by whether they reside on well-structured or unstructured regions of main chain. Open circles identify cases explained in the main text. Data can be found in Table S1.

Figure 2

Global unfolding on one face of the β-barrel. Standard colors and stick diagrams identify main chain atoms superimposed on secondary structural elements and connecting loops diagrammed as background. H-bond donors and acceptors are indicated. Log of protection factors are in colors as in Figure 1, indicating HX by way of unfolding (green), local fluctuations (red), or unknown (black). The red sphere is a crystallographically defined water molecule.

Figure 3

Heterogeneous exchange on the other face of the β-barrel. The identification scheme is as described for Figure 2.

Figure 4

Varied exchange mechanisms in the three α-helices. The identification scheme is as described for Figure 2.

Figure 5

Loop regions and defined water molecules. The identification scheme is as described for Figure 2. (A) The α1/β4 loop, (B) the α2/α3 loop, α1/β3 loop, and C-terminal region, and (C) the β4/β5 loop and near C-terminal region. Asn138 (B) was reoriented in NMR (2KQ3). Thr82 was involved in a crystal lattice contact (1SNO).

Scheme 1