Table 1.
Sequences (positions 61 to 67 for RnCD2*, 41–47 for ActP2*, and 15–21 for WW: positions where amino acids were perturbed appear in boldface), folding free energies, and folding and unfolding rate constant (k) ratios for RnCD2ad-, AcyP2- and Pin WW-derived glycoproteins and their non-glycosylated counterparts.†
| Protein | Sequence‡ | Δ Gf (kcal/mol) | ΔΔ Gf (kcal/mol) | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 61 | 67 | ||||||||||||||
| RnCD2* | -- | E | I | L | A | N | G | T | -- | −5.1 ± 0.4 | −0.6 ± 0.6 | 1.2 ± 0.1 | 0.5 ± 0.6|| | ||
| g-RnCD2* | -- | E | I | L | A | N | G | T | -- | −5.7 ± 0.5 | |||||
| RnCD2*-K | -- | K | I | L | A | N | G | T | -- | −4.0 ± 0.5 | −1.5 ± 0.6 | 1.5 ± 0.4 | 0.1 ± 0.1 | ||
| g-RnCD2*-K | -- | K | I | L | A | N | G | T | -- | −5.5 ± 0.4 | |||||
| RnCD2*-F | -- | E | I | F | A | N | G | T | -- | −4.2 ± 0.2 | −1.8 ± 0.4 | 1.0 ± 0.2 | 0.06 ± 0.01 | ||
| g-RnCD2*-F | -- | E | I | F | A | N | G | T | -- | −6.0 ± 0.3 | |||||
| RnCD2*-K,F | -- | K | I | F | A | N | G | T | -- | −2.1 ± 0.4 | −3.7 ± 0.6 | 2.4 ± 0.7 | 0.005 ± 0.004¶ | ||
| g-RnCD2*-K,F | -- | K | I | F | A | N | G | T | -- | −5.8 ± 0.5 | |||||
| 41 | 47 | ||||||||||||||
| AcyP2* | -- | K | N | T | A | N | G | T | -- | −4.1 ± 0.7 | 0.5 ± 0.8 | N.D.§ | N.D. | ||
| g-AcyP2* | -- | K | N | T | A | N | G | T | -- | −3.6 ± 0.4 | |||||
| AcyP2*-F | -- | K | N | F | A | N | G | T | -- | −2.5 ± 0.5 | −2.0 ± 0.7 | N.D. | N.D. | ||
| g-AcyP2*-F | -- | K | N | F | A | N | G | T | -- | −4.5 ± 0.8 | |||||
| 15 | 21 | ||||||||||||||
| WW | -- | M | S | R | S | N | G | R | -- | −0.11 ± 0.05 | 0.26 ± 0.06 | 0.70 ± 0.06 | 1.0 ± 0.1 | ||
| g-WW | -- | M | S | R | S | N | G | R | -- | 0.15 ± 0.04 | |||||
| WW-F | -- | M | F | R | S | N | G | R | -- | 0.29 ± 0.04 | 0.07 ± 0.07 | 0.89 ± 0.09 | 1.0 ± 0.1 | ||
| g-WW-F | -- | M | F | R | S | N | G | R | -- | 0.36 ± 0.05 | |||||
| WW-T | -- | M | S | R | S | N | G | T | -- | 0.19 ± 0.06 | 0.08 ± 0.08 | 0.86 ± 0.07 | 1.0 ± 0.1 | ||
| g-WW-T | -- | M | S | R | S | N | G | T | -- | 0.28 ± 0.05 | |||||
| WW-F,T | -- | M | F | R | S | N | G | T | -- | 0.69 ± 0.05 | −0.70 ± 0.08 | 2.0 ± 0.3 | 0.7 ± 0.1 | ||
| g-WW-F,T | -- | M | F | R | S | N | G | T | -- | −0.01 ± 0.06 | |||||
Tabulated data are given as mean ± standard error at 25 °C for RnCD2* and AcyP2*, and are calculated at 55 °C for WW variants. For tabulated rate constants, see Fig. S3 and Table S3 (18).
N= Asn(glycan). The glycan is mostly Man6–8 oligomannose for RnCD2* variants, is mostly fucosylated paucimannose for AcyP2* variants, and is GlcNAc for WW variants (see Table S1) (18).
N.D., not determined.
This unfolding rate ratio is within error of 1, indicating that the unfolding rates are not significantly different. It is also within error of being negative; this is an artifact of treating this naturally positive quantity as being normally distributed.