TABLE 1.

Activity, autoprocessing, CD spectroscopy, and MALLS analysis on GPR variants^a

GPR	Result for:
	P41			P46
	SASP assayb	Folded by CDc	MALLS (T:D:M)d	Autoprocessinge	Folded by CD	MALLS (T:D:M)
Wild type	Active	Folded	60:0:0	Active	Folded	90:0:0
Inactive single variants
D127A	Inactive	Folded	80:0:0	Inactive	Folded	100:0:0
D127E	Inactive	Folded	80:0:0	Inactive	Folded	100:0:0
D127N	Inactive	Folded	0:40:0	Inactive	Folded	90:0:0
D127S	Inactive	Folded	0:20:0	Inactive	Folded	90:0:0
D193N	Inactive	Folded	80:0:0	Inactive	Folded	90:0:0
Active single variants
Group Ag	Active	NDf	ND	Active	Folded	ND
Group Bh	Active	Folded	ND	Active	Folded	ND
S199A	Active	Folded	10:30:10	Inactive	Folded	80:0:0
R202Q	Active	Folded	ND	Active	Folded	90:0:0
D246N	Partially active	Folded	Aggregatedi	Inactive	Folded	Aggregated
S249A	Partially active	Folded	Aggregated	Inactive	Folded	Aggregated
Deletion variants
Δ223	Active	Folded	ND	Active	Folded	ND
Δ213-226	Inactive	Misfolded	Aggregated	Inactive/inactive	Misfolded	Aggregated
Δ213-227	Inactive	Misfolded	Aggregated	Inactive/inactive	Misfolded	Aggregated
Δ214-226	Inactive	Misfolded	ND	Inactive/inactive	Misfolded	ND
Δ214-227	Inactive	Misfolded	ND	Inactive/inactive	Misfolded	ND

^aAssays of activity, autoprocessing, CD spectroscopy, and MALLS on wild-type P₄₁ and P₄₆ and all variants were done as described in Materials and Methods.

^bActive, no significant difference (± 20%) in activity relative to that of wild-type P₄₁. Inactive, at least 80-fold less active than wild-type P₄₁. Partially active, ≈50% activity relative to that of wild-type P₄₁.

^cFolded, no significant difference between a variant's CD spectrum and that of the corresponding wild-type GPR (P₄₁ or P₄₆) (see Fig. 3). Misfolded, significant difference between a variant's CD spectrum and that of wild-type GPR.

^dData are reported as ratios of the percent tetramer (T) to dimer (D) to monomer (M). These values may not add up to 100% because most proteins did not completely give well-defined populations of homogeneous particle size. (See Fig. 4.)

^eActive, generation of P₄₁ after overnight incubation under autoprocessing conditions. Inactive, no P₄₁ generated. Wild-type P₄₆ typically autoprocesses to 50% completion overnight, and the sensitivity of this assay allows detection of ≈3% autoprocessing. Inactive/inactive, P₄₆ variant was unable to autoprocess to P₄₁ and was inactive against SASP as P₄₆.

^fND, not done.

^gD256N, E26Q, E89Q, H215Q, K21P, Q208E, S212A, S218A, T74D, T215V, and Y72F.

^hD153N, D211N, D253N, E201Q, H142Q, K223A, K223E, K223H, K223M, S210A, R224Q, T125N, and T173V.

ⁱAggregated, MALLS analysis yielded only high-molecular-weight species with no detectable tetramer, dimer, or monomer.