VOLUME 283 (2008) PAGES 30950–30956
During routine maintenance of DNA plasmid stocks in our laboratory, we discovered that one of the mutant proteins characterized previously (Baden, E. M., Owen, B. A., Peterson, F. C., Volkman, B. F., Ramirez-Alvarado, M., and Thompson, J. R. (2008) J. Biol. Chem. 283, 15853–15860) did not have the correct amino acid sequence. An error during the initial sequence analysis resulted in the incorrect identification of the triple reciprocal mutant. The protein that was identified in the publication as κI O18/O8 N34I/K42Q/Y87H actually lacked the K42Q mutation. This error does not alter the overall conclusions of the work. All other proteins were confirmed to have the proper sequence by reviewing mass spectrometry and DNA sequence analysis results. We apologize for any inconvenience or misunderstanding that this error may have caused.
To provide the information we initially sought to include in the publication, we carried out site-directed mutagenesis and successfully expressed the κI O18/O8 N34I/K42Q/Y87H mutant protein. The sequence of this protein was verified by mass spectrometry peptide analysis. The methods for all experiments described below are detailed in the original publication.
A thermodynamic characterization of κI O18/O8 N34I/K42Q/Y87H revealed that the protein's thermodynamic stability is very similar to that of the double mutant κI O18/O8 N34I/Y87H and to that of AL-09, the amyloidogenic protein discussed in the article. Thermal denaturation of κI O18/O8 N34I/K42Q/Y87H resulted in a Tm (melting temperature, where half of the protein is unfolded) of 38.4 °C (Table 1). The ΔGfolding value is also among the least favorable of the mutants, as is the Cm value derived from the urea denaturation (Table 1).
TABLE 1.
Thermodynamics of restorative and reciprocal mutants
Error is the S.D. of at least three independent experiments. Proteins are in order from least to most favorable ΔGfolding.
| Protein | Tm | TmNaS | Cm | ΔGfolding | ΔH |
|---|---|---|---|---|---|
| °C | °C | m | kcal/mol | kcal/mol | |
| AL-09a | 41.1 ± 1.0 | 50.4 ± 0.6 | 1.88 ± 0.07 | −3.53 ± 0.28 | −62.8 ± 1.0 |
| κI O18/O8 N34I/Y87H | 39.5 ± 1.0 | 50.8 ± 0.3 | 1.89 ± 0.06 | −3.81 ± 0.66 | −79.7 ± 4.2 |
| AL-09 Q42K | 40.2 ± 0.3 | 50.4 ± 0.3 | 1.80 ± 0.25 | −4.20 ± 0.86 | −75.3 ± 1.8 |
| κI O18/O8 Y87H | 47.3 ± 0.4 | 59.6 ± 0.7 | 2.98 ± 0.28 | −4.58 ± 0.38 | −77.3 ± 3.5 |
| κI O18/O8 N34I/K42Q/Y87H | 38.4 ± 1.9 | 47.6 ± 0.6 | 1.90 ± 0.10 | −4.41 ± 0.77 | −61.4 ± 7.8 |
| AL-09 I34N | 48.6 ± 0.2 | 58.8 ± 0.3 | 2.92 ± 0.22 | −5.34 ± 0.72 | −77.3 ± 2.4 |
| AL-09 H87Y | 54.6 ± 0.6 | 64.3 ± 0.5 | 3.29 ± 0.04 | −6.10 ± 0.30 | −100.1 ± 10.3 |
| κI O18/O8a | 56.1 ± 0.2 | 68.0 ± 0.3 | 3.98 ± 0.07 | −6.12 ± 0.23 | −95.7 ± 2.6 |
| AL-09 I34N/H87Y | 58.0 ± 0.1 | 69.6 ± 0.2 | 4.52 ± 0.16 | −6.84 ± 1.27 | −100.5 ± 7.4 |
| κI O18/O8 N34I | 51.5 ± 0.9 | 65.3 ± 0.3 | 3.06 ± 0.17 | −7.17 ± 1.50 | −109.5 ± 8.5 |
a Data were reported previously (Baden, E. M., Owen, B. A., Peterson, F. C., Volkman, B. F., Ramirez-Alvarado, M., and Thompson, J. R. (2008) J. Biol. Chem. 283, 15853–15860).
In addition to the thermodynamic parameters, we made several qualitative observations that the κI O18/O8 N34I/K42Q/Y87H protein appears to be the least stable of all the mutants. The far-UV CD spectrum, which verifies the secondary β-sheet structure of the protein, is noisier than that for any other protein we have studied, and the minima at 218 and 235 nm are less pronounced. The protein also appears to decrease in thermodynamic stability daily when stored at 4 °C.
We also analyzed the fibril formation properties of the κI O18/O8 N34I/K42Q/Y87H mutant. Our previous experimental procedure included incubating 20 mm protein and 500 mm Na2SO4 at the TmNaS of the protein for ∼5 days to form fibrils that would then be used to seed a fibril formation reaction at 37 °C. For the κI O18/O8 N34I/K42Q/Y87H protein, however, even 12 days of incubation did not produce much thioflavin T fluorescence enhancement to indicate that fibrils had formed. Doubling the protein concentration to 40 μm did not increase the amount of fluorescence enhancement. Electron microscopy revealed that fibrils were produced in these reactions, but they were extraordinarily tiny, accounting for the limited thioflavin T fluorescence. The morphology of the fibrils also differed from that of fibrils formed by the other proteins in that the fibril edges were not smooth and straight. Coupled with the thermodynamic observations, these results may indicate that too much of the protein was already unfolded and could not provide the structure needed to form normal fibrils. The size and unusual morphology of the κI O18/O8 N34I/K42Q/Y87H fibrils precluded us from carrying out the seeded fibril formation at 37 °C, as we were able to do with the other mutants described in the article.
Collectively, the data from the κI O18/O8 N34I/K42Q/Y87H mutant suggest that the combination of the three nonconservative mutations in the amyloidogenic protein reduces the stability and may act in concert to induce amyloidogenesis when introduced into the germ-line protein.