Evidence, from simulations, of a single state with residual native structure at the thermal denaturation midpoint of a small globular protein

Abstract

The folding of the B-domain of staphylococcal protein A has been studied by coarse-grained canonical and multiplexed replica-exchange molecular dynamics simulations with the UNRES force field in a broad range of temperatures (270 K < or = T < or = 350 K). In canonical simulations, the folding was found to occur either directly to the native state or through kinetic traps, mainly the topological mirror image of the native three-helix bundle. The latter folding scenario was observed more frequently at low temperatures. With increase of temperature, the frequency of the transitions between the folded and misfolded/unfolded states increased and the folded state became more diffuse with conformations exhibiting increased root-mean-square deviations from the experimental structure (from about 4 A at T = 300 K to 8.7 A at T = 325 K). An analysis of the equilibrium conformational ensemble determined from multiplexed replica exchange simulations at the folding-transition temperature (T(f) = 325 K) showed that the conformational ensemble at this temperature is a collection of conformations with residual secondary structures, which possess native or near-native clusters of nonpolar residues in place, and not a 50-50% mixture of fully folded and fully unfolded conformations. These findings contradict the quasi-chemical picture of two- or multistate protein folding, which assumes an equilibrium between the folded, unfolded, and intermediate states, with equilibrium shifting with temperature but with the native conformations remaining essentially unchanged. Our results also suggest that long-range hydrophobic contacts are the essential factor to keep the structure of a protein thermally stable.

Figure 1

Heat-capacity (solid line), ensemble-averaged radius of gyration (long-dashed line) and ensemble-averaged RMSD (short-dashed line) of 1BDD, calculated in MREMD simulations.

Figure 2

Scheme of the top view of (a) native and (b) mirror-image topology of the three-helix bundle of protein A. Each helix is represented by a circle with the number identifying its position in the chain (1 – N-terminal, 2 – middle, 3 – C-terminal) and lines showing connections between the helices. The C-terminal α-helix runs from bottom to top.

Figure 3

Structures and FEPs of pathways corresponding to (a) folding through a kinetic trap, with such pathways occurring most frequently at low temperatures (T = 270K and 280K); (b) downhill folding, with such pathways occurring near (310K) but below the folding-transition temperature (T = 325K); and (c) residual folding, with such pathways occurring at the folding-transition temperature (T = 325K). The structures are colored from blue to red from the N-to the C-terminus. A-5, A-7 and A-9 are representative structures of a kinetic trap (mirror image), the transition state and the native state, respectively. B-6 and C-3 correspond to native and residually-folded structures, respectively.

Figure 4

Experimental structure of 1BDD (a) and four representatives from the canonical ensemble determined by MREMD simulations at T = 325 K (b-e) with nonpolar residues (Ala13, Phe14, Ile17, Leu18, Phe31, Ile32, Leu35, Leu45, Leu46, Ala49, and Leu52) that make contact between helices in the N-terminal and the middle or the N-terminal and the C-terminal α-helix in the experimental structure and in the conformations of the ensemble at T = 325 K, shown as bonds between heavy atoms for the experimental structure or as spheres at the united side-chain centers for the UNRES structures. The residues of the N-terminal, middle, and C-terminal helices are colored blue, green, and red, respectively. Native contacts between the above-mentioned nonpolar residues are shown as black dashed lines, and non-native contacts (in panels b-e) are shown as pink dashed lines. It is shown that a cluster composed of all or some of the above-mentioned nonpolar residues is preserved regardless of the formation of secondary structure or topology which is similar to that of the native structure in conformations (b) and (c) or mirror image (d) and (e). Additionally, in the structures shown in panel (b) and (d), the neighboring nonpolar residues that are not involved in the formation of long-range contacts in the experimental structure: Ala43 and Tyr15, respectively, can assist in the formation of long-range contacts between the N-terminal, middle, and C-terminal segments of the chain.

Figure 5

Distributions of C^α rmsd from the NMR structure of 1BDD, at (a) T = 300 K, (b) T = 315 K, (c) T=325 K (T_f), and (d) T = 350 K, of the conformations with native (blue) and mirror-image (red) topology of the three-helix bundle. The fractions of the conformations with native (f_N) and mirror-image bundle topology (f_M) are shown in the respective panels. The bin size is Δd = 0.5 Å.

Figure 6

Plots of the probability of states at T = 300 K (solid lines), T = 330 K (short-dashed lines) and T=350 K (dotted lines) as a function of (a) the order parameter q; (bin size Δq=0.1) and (b) the UNRES energy (U; bin size ΔU=5 kcal/mol).

Figure 7

(a) Map of the side chain – side chain (right triangle) and side backbone peptide-group-hydrogen-bonding contacts (left triangle) of the experimental structure of 1BDD. (b) – (d) Probability maps of these contacts corresponding to the conformational ensembles at (b) T = 300 K, (c) T = 325 K, and (d) T = 350 K. The probability color scale is shown in the upper panel.

Figure 8

Plots of the ensemble-averaged number of 1,4-backbone-hydrogen-bonding contacts (characteristic of α-helix; solid line; inflection point at T_i = 315 K, 32% of maximum number of contacts at T_i), 1,4-side-chain contacts (long-dashed line; T_i = 320 K, 50% of the maximum number of contacts at T_i), all long-range side-chain contacts (short-dashed line; T_i = 330K, 73% of the maximum number of contacts at T_i), and the contacts between the selected hydrophobic side chains that form the hydrophobic core and are labeled in Figure 4 (dot-dashed lines; T_i = 330 K, 78% of the maximum number of contacts at T_i).

Figure 9

Histograms of the end-to-end distance distribution (a) and the distributions of the distances between the C^α atoms of Gln10 and Ala55 (b) at T=300, 315, 325, and 350 K. Colors correspond to temperatures, as defined in the insets. The bin size is Δd=2 Å.