Probing the folding free energy landscape of the Src-SH3 protein domain

Abstract

The mechanism and thermodynamics of folding of the Src homology 3 (SH3) protein domain are characterized at an atomic level through molecular dynamics with importance sampling. This methodology enables the construction of the folding free energy landscape of the protein as a function of representative reaction coordinates. We observe that folding proceeds in a downhill manner under native conditions, with early compaction and structure formation in the hydrophobic sheet consisting of the three central beta strands of the protein. This state bears considerable resemblance to the experimentally determined transition state for folding. Folding proceeds further with the formation of the second hydrophobic sheet consisting of the terminal strands and the RT loop. The final stages of folding appear to involve the formation of the hydrophobic core through the expulsion of water molecules bridging the two hydrophobic sheets. This work sheds new light on the complementary roles of sequence and topology in governing the folding mechanism of small proteins and provides further support for the role of water in facilitating the late stages in folding.

Fig 1.

(a) Ribbon diagram of src-SH3 (Protein Data Bank ID code ) and native contact probability map. A native contact between non-neighboring residues was considered to be formed if the centers of geometry of the side chains were within 6.5 Å of each other. (b) Contact probability map for ρ = 0.2 (above diagonal line) and ρ = 0.4 (below diagonal line). At ρ = 0.2, early secondary structure appears in and between strands β2 and β3. By ρ = 0.4, the central three-stranded sheet consisting of strands β2, β3, and β4 has formed (with probabilities near 1 between strands β2 and β3, and greater than 0.6 between strands β3 and β4). The rest of the protein is mostly unstructured. (c) Contact probability map for ρ = 0.6 (above diagonal line) and ρ = 0.8 (below diagonal line). By ρ = 0.6, the protein is nearly as compact as in the native state, and the central three-stranded β2-β3-β4 sheet has fully consolidated. Structure has begun to form in the second hydrophobic sheet consisting of a portion of the RT loop and the two terminal strands. The two hydrophobic sheets do not make native contacts with each other. By ρ = 0.8, the protein is nearly native, with both hydrophobic sheets formed and mostly packed.

Fig 2.

(a) pmf at 298 K as a function of ρ. The free energy surface is downhill with a native basin at r ≥ 0.8. The stability of the native state is ≈3 kcal/mol. (b) pmf at 298 K as a function of ρ and radius of gyration, R_g (in angstroms). Contours are drawn every 1 kcal/mol. (c) pmf at 298 K as a function of ρ and the number of water molecules in the core. Contours are drawn every 0.6 kcal/mol. (d) Three high-temperature unfolding runs (T = 400 K) superimposed onto the free energy surface at 298 K.