Structural study of hNck2 SH3 domain protein in solution by circular dichroism and X-ray solution scattering.

We have done conformational study of hNck2 SH3 domain by means of far-ultraviolet (far-UV) circular dichroism (CD) and X-ray solution scattering (XSS). The results indicated that the following: (1) hNck2 SH3 domain protein exhibited concentration dependent monomer-dimer transition at neutral pH, while the secondary structure of this protein was independent of the protein concentration. (2) The hNck2 SH3 domain also exhibited pH dependent monomer-dimer transition. This monomer-dimer transition was accompanied with helix-ß transition of the secondary structural change. Moreover, the acid-induced conformation, which was previously studied by Liu and Song by CD and nuclear magnetic resonance (NMR), was found to be not compact, but the conformation of the protein at acidic pH was similar to the cold denatured state (C-state) reported by Yamada et al. for equine ß-lactoglobulin. We calculated that a structure of the equilibrium helix-rich intermediate of the hNck2 SH3 domain by DAMMIF program.

Transient helical structure during PI3K and Fyn SH3 domain folding.

A growing list of proteins, including the ß-sheet-rich SH3 domain, is known to transiently populate a compact α-helical intermediate before settling into the native structure. Examples have been discovered in cryogenic solvent as well as by pressure jumps. Earlier studies of λ repressor mutants showed that transient states with excess helix are robust in an all-α protein. Here we extend a previous study of src SH3 domain to two new SH3 sequences, phosphatidylinositol 3-kinase (PI3K) and a Fyn mutant, to see how robust such helix-rich transients are to sequence variations in this ß-sheet fold. We quantify helical structure by circular dichroism (CD), protein compactness by small-angle X-ray scattering (SAXS), and transient helical populations by cryo-stopped-flow CD. Our results show that transient compact helix-rich intermediates are easily accessible on the folding landscape of different SH3 domains. In molecular dynamics simulations, force field errors are often blamed for transient non-native structure. We suggest that experimental examples of very fast α-rich transient misfolding could become a more subtle test for further force field improvements than observation of the native state alone.

A stable alpha-helix-rich intermediate is formed by a single mutation of the beta-sheet protein, src SH3, at pH 3.

Recently, we have found a transient intermediate on the folding pathway of src SH3. Intending to investigate the structure of the transient intermediate, we tested a mutant of src SH3, named A45G, using circular dichroism, fluorescence and X-ray solution scattering, and incidentally found that it forms a stable alpha-helix-rich intermediate (I(eq)) (different from the native beta-sheet-based secondary structure) at pH 3.0, but contains only beta-sheets at pH 6.0, whereas wild-type SH3 forms only beta-sheets at both pH 3.0 and pH 6.0. The intermediate I(eq) shows a circular dichroism measured at theta(222)=-10,300 deg.cm(2) dmol(-1), indicating a 31% alpha-helix proportion, as estimated by the CONTIN program. X-ray scattering gave the radius of gyration for I(eq) as 19.1 A at pH 3.0 and 15.4 A at pH 6.0, and Kratky plots showed a clear peak at pH 3.0, 4.0 and 6.0, indicating that I(eq) too is compact. In these parameters, I(eq) closely resembles the kinetically-obtained intermediate I(kin) which we found on the folding pathway of wild-type SH3 at pH 3.0 (radius of gyration 18.7 A and theta(222)=-8700 deg.cm(2)dmol(-1)), indicating a 26% alpha-helix proportion in our previous paper. Refolding experiments with A45G were done at pH 6.0 by stopped-flow apparatus monitored by circular dichroism, and compared to kinetic experiments with wild-type SH3 at pH 6.0. The result showed an alpha-helix-rich intermediate at the same dichroism amplitude, but nine times slower in formation-rate. A pH-jump experiment from pH 3.0 to pH 5.9 on A45G was also performed. This showed no bursts, and the rate of conformation-change was almost as fast as the refolding rate of A45G at pH 6.0. These kinetic experiment data would be consistent with I(eq) being nearly identical to the I(kin), which appeared on the folding pathways of both wild-type SH3 and A45G at pH 3.