Nanopore analysis reveals differences in structural stability of ovine PrP^C proteins corresponding to scrapie susceptible (VRQ) and resistance (ARR) genotypes

Figures & data

Figure 1. Representation and documentation of Type I and Type II events through nanopore analysis. (A) Cartoon representation of the interaction between the pore and protein of interest that lead to Type I and Type II (intercalation or translocation events). (B) Raw data from the current project illustrating Type I and Type II events.

Table 1. Event parameters for Ovine PrP(94–234), ARR genotype and Ovine PrP(94–233), VRQ genotype pre-treated with 0 M, 1 M, 2 M, and 3 M Gdn-HCl^a

Compound	I1 (pA)	I2 (pA)	T1 (ms)	T2 (ms)	A1	A1(%)	A2	A2(%)	A1/A2	W1	W2
OvPrP:ARR genotype, (0M Gdn-HCl)	-27.2 (0.1)	-	0.21 (0.03)	-	1712	76	551	24	3.10	2.7	-
OvPrP:ARR genotype, (1M Gdn-HCl)	-30.1 (0.6)	-	0.20 (0.05)	-	243	55	197	45	1.23	10.8	-
OvPrP:ARR genotype, (2M Gdn-HCl)	-30.0 (0.4)	-76.9(1.6)	0.19 (0.06)	0.09 (0.02)	728	49	757	51	0.96	8.0	20.0
OvPrP:ARR genotype, (3M Gdn-HCl)	-28.9 (0.2)	-58.5 (1.5)	0.06 (0.01)	0.10 (0.01)	550	53	495	47	1.1	9.2	30.0
OvPrP:VRQ genotype, (0M Gdn-HCl)	-26.4 (0.05)	-	0.02 (0.00)	-	461	65	247	35	1.86	4.0	-
OvPrP:VRQ genotype, (1M Gdn-HCl)	-29.6 (0.4)	-70.0 (0.0)	0.03 (0.01)	0.03 (0.01)	493	52	453	48	1.08	13.6	30.0
OvPrP:VRQ genotype, (2M Gdn-HCl)	-29.0 (0.2)	-82.0 (0.0)	0.17 (0.01)	0.16 (0.01)	412	37	708	63	0.58	6.5	30.0
OvPrP:VRQ genotype, (3M Gdn-HCl)	-30.4 (0.2)	-73.40 (1.2)	0.06 (0.01)	0.14 (0.01)	869	48	952	52	0.91	7.8	26.1

^aI and T represent the intensities and durations of the current blockades of the event populations presented in Figure 2. The subscripts 1 and 2 refer to Type I (bumping) and Type II (intercalation/translocation) event populations. A₁ and A₂ are the number of events of each population. W₁ and W₂ are the peak widths at half-height. (The values in parenthesis are the standard deviations).

Figure 2. Nanopore Analysis of Denaturation of Ovine ARR and VRQ PrP^C. Current blockade histograms for E.coli expressed sheep PrP^C for the two genotypes, ARR and VRQ. (A) under non-denaturing conditions (0 M Gdn-HCl): (a) OvPrP(94–234) ARR genotype, (b) OvPrP(94–233) VRQ genotype. (B) Under denaturing conditions (1 M Gdn-HCl): (c) OvPrP(94–234) ARR genotype, (d) OvPrP(94–233) VRQ genotype. (C) Under denaturing conditions (2 M Gdn-HCl): (e) OvPrP(94–234) ARR genotype, (f) OvPrP(94–233) VRQ genotype. (D) Under denaturing conditions (3 M Gdn-HCl): (g) OvPrP(94–234) ARR genotype, (h) OvPrP(94–233) VRQ genotype. The values are listed in Table 1.

Figure 3. Blockade time for molecules interacting with the α-hemolysin pore in nanopore analysis for Ovine ARR and VRQ PrP^C pre-treated in 2 M Gdn-HCl. Exponential curves depicting the blockade time for (A) bumping time (T1) for OvPrP(94–234) ARR genotype. (B) Intercalation/translocation time (T2) for OvPrP(94–234) ARR genotype. (C) bumping time (T1) for OvPrP(94–233) VRQ genotype. (D) Intercalation/translocation time (T2) for OvPrP(94–233) VRQ genotype.

Figure 4. Prion antibody interactions. Current blockade histograms for sheep PrP^C genotypes, ARR and VRQ pre-treated in 2M Gdn-HCl with YML or SN6b antibodies. (A) YML antibody interactions: (a) OvPrP(94–234), ARR genotype pre-treated in 2 M Gdn-HCl with YML at a 1:1 ratio. (b) OvPrP(94–233), VRQ genotype pre-treated in 2 M Gdn-HCl with YML at a 1:1 ratio. (c) Overlapping histograms of OvPrP(94–234), ARR genotype pre-treated in 2 M Gdn-HCl with and without YML antibody. (d) Overlapping histograms of OvPrP(94–233), VRQ genotype pre-treated in 2 M Gdn-HCl with and without YML antibody. (B) SN6b antibody interactions: (e) OvPrP(94–234), ARR genotype pre-treated in 2 M Gdn-HCl with SN6b at a 1:1 ratio. (f) OvPrP(94–233), VRQ genotype pre-treated in 2 M Gdn-HCl with SN6b at a 1:1 ratio. (g) Overlapping histograms of OvPrP(94–234), ARR genotype pre-treated in 2 M Gdn-HCl with and without SN6b antibody. (h) Overlapping histograms of OvPrP(94–233), VRQ genotype pre-treated in 2 M Gdn-HCl with and without SN6b antibody. The values are listed in Table 2.

Table 2. Event parameters for OvPrP(94–234):ARR genotype and OvPrP(94–233), VRQ genotype in 2M Gdn-HCl with YML or SN6b complex^a

Compound	I1 (pA)	I2 (pA)	T1 (ms)	T2 (ms)	A1	A1(%)	A2	A2(%)	A1/A2	W1	W2
OvPrP(94–234):ARR genotype (2M Gdn-HCl)/YML	-25.8 (0.6)	-	0.16 (0.01)	-	620	56	480	44	1.29	12.2	-
OvPrP(94–233):VRQ genotype (2M Gdn-HCl)/YML	-28.5 (0.2)	-61.9 (1.2)	0.11 (0.01)	0.22 (0.01)	665	51	627	49	1.06	8.6	30.2
OvPrP(94–234):ARR genotype (2M Gdn-HCl)/SN6b	-31.2 (0.2)	-66.8 (1.3)	0.03 (0.0)	0.06 (0.01)	422	45	522	55	0.81	7.9	30.4
OvPrP(94–233):VRQ genotype (2M Gdn-HCl)/SN6b	-26.5 (0.1)	-	0.25 (0.05)	-	645	65	342	35	1.88	3.0	-

^aI and T represent the intensities and durations of the current blockades of the event populations presented in Figure 4. The subscripts 1 and 2 refer to Type I (bumping) and Type II (intercalation/translocation) event populations. A₁ and A₂ are the number of events of each population. W₁ and W₂ are the peak widths at half-height. (The values in parentheses are the standard deviations).