Masaji Shinjo

Transient Helical Structure during PI3K and Fyn SH3 Domain Folding

A growing list of proteins, including the β-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 β-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.

α-Helical burst on the folding pathway of FHA domains from Rad53 and Ki67

We investigated refolding processes of beta-sheeted protein FHA domains (FHA1 domain of Rad53 and Ki67 FHA domain) by cryo-stopped-flow (SF) method combined with far-ultraviolet (far-UV) circular dichroism (CD, the average secondary structure content) and small angle X-ray scattering (SAXS, measuring the radius of gyration). In case of FHA1 domain of Rad53, no detectable time course was observed except the initial burst on its refolding process at 4 degrees C, suggesting that the FHA1 domain of Rad53 was already refolded to its native state within the dead time of the SF apparatus and the rate of the refolding is too fast to be observed at this temperature. In contrast, there was an observable alpha-helical burst at -15 degrees C and -20 degrees C in the presence of 45% ethylene glycol (EGOH) by CD-SF. Besides, the radius of gyration (Rg) of the burst phase intermediate at -20 degrees C shows the intermediate is already compact, and the compaction process was accompanied with the decrease of alpha-helical content at the same temperature. In case of Ki67 FHA domain, ellipticity change at 222 nm was observed on its refolding pathway at -28 degrees C in the presence of 45% EGOH and 2 mM DTT, indicating that Ki67 FHA domain also takes non-native alpha-helix-rich intermediate on its folding pathway. Time-resolved SAXS experiment was done. As the signal/noise ratio is low, we could not observe the time-dependent signal change through the time course. However, the initial Rg value was obtained as 18.2 +/- 0.5 A, which is much smaller than the unfolded Rg value (26.5 +/- 1.2 A), and is slightly larger than the native one (15.9 +/- 1.8 A). These results suggest that Ki67 FHA domain also forms compact non-native alpha-helix-rich intermediate before refolding to its native beta-structure on the refolding pathway. These results are in good agreement with other beta-proteins, such as bovine beta-lactoglobulin (BLG), src SH3 domain proteins. It seems the alpha-helical burst phases appear on the folding pathway of beta-sandwiched proteins.

α-helix formation rate of oligopeptides at subzero temperatures.

In 1999, Clarke et al. ((1999) Proc. Natl. Acad. Sci. USA 96, 7232–7237) reported that the nucleation rate of α-helix of oligopeptide AK16 is as slow as 60 ms. In the present study, we measured the nucleation rate of oligopeptide, C17 (DLTDDIMCVKKILDKVG, corresponding to α-helical region of 84th to 100th amino acids of bovine α-lactalbumin) using the same method as Clarke et al. We found only initial bursts of the increase of α-helices at temperatures higher than −50°C in the presence of 70% methanol. The result with AK16 was the same as Clarke et al. reported. We also found that the folding rate of polyglutamic acid is too fast to be detected by the stopped-flow apparatus at 4°C. These results demonstrate that the α-helix formation rates in C17, AK16 and polyglutamic acid are shorter than the dead time of the stopped-flow apparatus (6 ms).

Structural stability of E. coli trigger factor studied by synchrotron small-angle X-ray scattering

Solution small-angle X-ray scattering (SAXS) is an effective technique for quantitatively measuring the compactness and shape of proteins. We use SAXS to study the structural characteristics and unfolding transitions induced by urea for full length Escherichia coli trigger factor (TF) and a series of truncation mutants, obtaining and comparing the radiuses of gyration (Rg), the distance-distribution function (P(r) function) and integrated intensity of TF variants in native and unfolding states. The C-terminal 72-residue truncated mutant TF360 exhibited dramatic structural differences and reduced stability compared with the whole TF molecule, while the N-domain truncated mutant MC maintained its compact structure with reduced stability. These results indicate that the C-terminal region of TF plays an important role in the structural and conformational stabilities of the TF molecule, while the N-domain is relatively independent.

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-β 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 β-lactoglobulin. We calculated that a structure of the equilibrium helix-rich intermediate of the hNck2 SH3 domain by DAMMIF program.