Table 2.

Kinetic parameters obtained from fitting stopped-flow kinetics data to a three-state model for unfolding

Parametera,b	WT GlpG	V211C	Cys-less (C104V)	V260C
log kfno SDS	−0.37 ± 0.33	−1.70 ± 0.21	0.07 ± 1.61	−0.40 ± 0.07
mf (M−1)	−3.99 ± 0.75	−1.07 ± 0.52	−5.74 ± 3.65	−5.42 ± 0.33
log KIno SDS	1.94 ± 0.31	0.78 ± 0.20	2.37 ± 1.58	≡ 2c
mI (M−1)	−7.11 ± 0.48	−7.45 ± 2.31	−6.51 ± 3.03	≡ −7c
log kuno SDS	−5.74 ± 0.18	−6.01 ± 0.33	−5.59 ± 0.20	−4.99 ± 0.17
mu (M−1)	5.85 ± 0.22	6.05 ± 0.38	6.02 ± 0.25	5.42 ± 0.22
log KD-NnoSDSd	−5.37 ± 0.38 (−5.43 ± 0.15e)	−4.30 ± 0.39	−5.66 ± 1.63	−4.59 ± 0.18
mD-Nf	9.84 ± 0.79 (−9.04 ± 0.56e)	7.12 ± 0.65	11.77 ± 3.66	10.84 ± 0.40
log KD-NnoSDSf	−8.50 ± 1.25	−5.53 ± 0.27	−7.37 ± 0.81	−7.17 ± 0.50
mD-Ne	14.85 ± 2.18	9.63 ± 0.47	15.22 ± 1.67	14.11 ± 0.99

^aAll data measured at 25°C in buffer B (360 mM NaCl, 1 mM EDTA, and 25 mM MOPS (pH 7.0)).

^bDefined in Eq. S3 describing the folding model C ↔ D ↔ N.

^cVery weak downturn made it unfeasible to determine these parameters. Instead, they were locked to values similar to those of the three other mutants.

^dCalculated as log K_D-N^{no SDS} = log k_u^{no SDS} − log k_f^{no SDS} and m_D-N = m_u − m_f.

^eFrom previous stopped-flow kinetics work on WT GlpG (18).

^fObtained from equilibrium denaturation data (Table 1). Included for comparison.