Table 1.

Apparent destabilization energies (ΔΔG) at the mitochondrial surface and in free solution in kcal/mol

Mutation in barnase	Destabilization at the mitochondrial surface*†	Destabilization of native state in free solution*‡	Acceleration of unfolding in free solution§¶	Destabilization of the molten globule state in free solution¶
Ile4 → Ala	0.6  ± 0.1	0.9  ± 0.1	1.5	0.0
His18 → Gln	0.7  ± 0.1	1.2  ± 0.1	0.2	1.2
Asn23 → Ala	1.4  ± 0.1	1.8  ± 0.1	2.6	−0.1
Ile25 → Val	0.7  ± 0.1	1.1  ± 0.1	1.5	−0.3
Thr26 → Ala	1.0  ± 0.2	1.9  ± 0.1	2.0	0.0
Val36 → Ala	0.6  ± 0.1	1.1  ± 0.1	1.5	−0.3
Ile51 → Val	1.1  ± 0.2	1.7  ± 0.1	2.2	−0.3
Asp54 → Ala	1.6  ± 0.2	2.6  ± 0.1	3.6	−0.5
Asp54 → Asn	1.4  ± 0.2	2.2  ± 0.1	3.0	−0.5
Asn58 → Ala	1.5  ± 0.1	2.1  ± 0.1	0.1	1.9
Asn77 → Ala	1.3  ± 0.2	1.8  ± 0.1	1.9	0.0

^*Mutations in barnase precursors. Mutations were generated in an Ile⁷⁶ → Val + Ile⁸⁸ → Val + Ile⁹⁶ → Val mutant background. Errors are SE calculated from at least three repeat measurements. 

^† The effects of mutations on protein stability at the mitochondrial surface were determined as the effects of mutations on import under the conditions used in these experiments. The apparent destabilization ΔΔG is calculated according to equation 4. Import experiments were performed at 35°C. 

^‡ Determined at 25°C in the import buffer lacking BSA. Results are the same at 35°C, within error. 

^§ We define acceleration of unfolding in free solution as ΔΔG_unfolding = RTln(k_mutant^unf/k_{wild type}^unf). 

^¶ Determined at 25°C in MES buffer (pH 6.3), data calculated from ref. 38. SE are smaller than 0.2 kcal/mol. Measurements were performed with barnase lacking a targeting sequence and lacking the mutant background.