Structural Details, Pathways, and Energetics of Unfolding Apomyoglobin

Abstract

Protein folding is often difficult to characterize experimentally because of the transience of intermediate states, and the complexity of the proteinsolvent system. Atomistic simulations, which could provide more detailed information, have had to employ highly simplified models or high temperatures, to cope with the long time scales of unfolding; direct simulation of folding is even more problematic. We report a fully atomistic simulation of the acid-induced unfolding of apomyoglobin in which the protonation of acidic side-chains to simulate low pH is sufficient to induce unfolding at room temperature with no added biasing forces or other unusual conditions; and the trajectory is validated by comparison to experimental characterization of intermediate states. Novel insights provided by their analysis include: characterization of a dry swollen globule state forming a barrier to initial unfolding or final folding; observation of cooperativity in secondary and tertiary structure formation and its explanation in terms of dielectric environments; and structural details of the intermediate and the completely unfolded states. These insights involve time scales and levels of structural detail that are presently beyond the range of experiment, but come within reach through the simulation methods described here. An implicit solvation model is used to analyze the energetics of protein folding at various pH and ionic strength values, and a reasonable estimate of folding free energy is obtained. Electrostatic interactions are found to disfavor folding.

Molten Globule Formation in Apomyoglobin Monitored by the Fluorescent Probe Nile Red

“…The corresponding local rmsd plot per residue (Supporting Information, Figure S3) indicates that the variation is due to a spatial movement more than to a local unfolding, which mainly concerns the connection loops between helices. The relative helicity for this structure is 0.75, which is in very good agreement with the experimental value of 0.77 (9). The variation with time of the backbone rmsd of the total structure and solvent-accessible surface (SASA) (Supporting Information, Figure S4) shows the convergence of the simulation.…”

“…Although the A, G, and H helices form a distinct tight subdomain in Mb, these helices are unstable when isolated as separate fragments (6,7). At pH 4, ApoMb adopts a conformation (I 4 ) that has been the subject of numerous structural, thermodynamic, kinetic, and theoretical studies (5,8,9). The I 4 conformation shows a decreased helix content and lacks the tight side-chain packing characteristic of the cores of globular proteins (6).…”

Exploring protein native states and large‐scale conformational changes with a modified generalized born model

“…25 To address this issue in optimizing parameters {␣, ␤, ␥} of GB OBC , we use snapshots from MD simulations that describe global conformational changes, such as the complete unfolding of a protein. The fit to the PB model has been performed on an MD trajectory that describes acid-unfolding of apomyoglobin, and includes both native and completely unfolded states at 300 K. 31 The latter has twice the size of the native state and about 20% residual helical structure, in agreement with the experiment. 32 At this point, we seek best agreement between the GB and PB models in computing the change in ⌬G el in going from the native (N) to unfolded (U) ensembles of structures (see Table I).…”

“…31 The native state is represented by 50 consecutive snapshots (2 ps apart from each other) with near-native radius of gyration, ϳ 16 Å, taken from the beginning of the acid-unfolding simulation. The unfolded state is represented by 50 consecutive snapshots from the end of that simulation, at which point the radius of gyration has approached ϳ30 Å-as is experimentally observed 32 in the unfolded state.…”

Partially Assembled Nucleosome Structures at Atomic Detail

“…Indeed, the wrapping of the nucleosomal DNA around the intact core histones is extremely tight-the free energy cost of complete DNA unwrapping is~40 kcal/mol (75), which is much larger than a stability of a typical protein (88,89). It is therefore not surprising that the question of how Pol II machinery overcomes this barrier to access the DNA has remained a fundamental open question in biology (25,90).…”