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1I5H

Chemical Shift Assignments and Coupling Constants for the rat Nedd4 WWIII domain - rat ENaC bP2 Peptide Complex

Authors

Kanelis, V., Rotin, D., Forman-Kay, J.D.

Assembly

Rat Nedd4 WWIII domain - ENaC bP2 Peptide Complex

Entity

1. rat Nedd4 WWIII domain (polymer, Thiol state: not present), 50 monomers, 5659.178 Da Detail

GSPVDSNDLG PLPPGWEERT HTDGRVFFIN HNIKKTQWED PRMQNVAITG

2. rat epithelial Na+ Channel, bP2 region (polymer, Thiol state: not present), 17 monomers, 1725.893 Da Detail

GSTLPIPGTP PPNYDSL

Total weight

7385.0713 Da

Max. entity weight

5659.178 Da

Source organism

Rattus norvegicus

Exptl. method

NMR

Data set

assigned_chemical_shifts, coupling_constants

Chem. Shift Complete1

Sequence coverage: 77.6 %, Completeness: 75.1 %, Completeness (bb): 75.7 % Detail

Polymer type: polypeptide(L)

	Total	¹H	¹³C	¹⁵N
All	75.1 % (574 of 764)	75.8 % (304 of 401)	73.4 % (218 of 297)	78.8 % (52 of 66)
Backbone	75.7 % (289 of 382)	77.9 % (102 of 131)	73.7 % (143 of 194)	77.2 % (44 of 57)
Sidechain	74.9 % (331 of 442)	74.8 % (202 of 270)	74.2 % (121 of 163)	88.9 % (8 of 9)
Aromatic	66.7 % (40 of 60)	66.7 % (20 of 30)	64.3 % (18 of 28)	100.0 % (2 of 2)
Methyl	79.3 % (46 of 58)	79.3 % (23 of 29)	79.3 % (23 of 29)

1. rat Nedd4 WWIII domain

GSPVDSNDLG PLPPGWEERT HTDGRVFFIN HNIKKTQWED PRMQNVAITG

2. rat epithelial Na+ Channel, bP2 region

GSTLPIPGTP PPNYDSL

Show sample condition

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

Chem. Shift Complete2

Sequence coverage: 35.8 %, Completeness: 51.3 %, Completeness (bb): 52.6 % Detail

Polymer type: polypeptide(L)

	Total	¹H	¹³C	¹⁵N
All	51.3 % (784 of 1528)	51.7 % (415 of 802)	50.5 % (300 of 594)	52.3 % (69 of 132)
Backbone	52.6 % (402 of 764)	53.4 % (140 of 262)	52.1 % (202 of 388)	52.6 % (60 of 114)
Sidechain	50.6 % (447 of 884)	50.9 % (275 of 540)	50.0 % (163 of 326)	50.0 % (9 of 18)
Aromatic	36.7 % (44 of 120)	36.7 % (22 of 60)	35.7 % (20 of 56)	50.0 % (2 of 4)
Methyl	54.3 % (63 of 116)	55.2 % (32 of 58)	53.4 % (31 of 58)

1. rat Nedd4 WWIII domain

GSPVDSNDLG PLPPGWEERT HTDGRVFFIN HNIKKTQWED PRMQNVAITG

2. rat epithelial Na+ Channel, bP2 region

GSTLPIPGTP PPNYDSL

Show sample condition

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

Coupling constant

89 J values in 4 lists
Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1) Detail

Release date

2001-04-29

Citation

Chemical Shift Assignments and Coupling Constants for the rat Nedd4 WWIII domain - rat ENaC bP2 Peptide Complex
Kanelis, V., Rotin, D., Forman-Kay, J.D.
Nat. Struct. Biol. (2001), 8, 407-412, PubMed , DOI:

Related entities 1. rat Nedd4 WWIII domain, : 120 entities Detail

Related entities 2. rat epithelial Na+ Channel, bP2 region, : 1 : 9 : 6 entities Detail

Interaction partners 1. rat Nedd4 WWIII domain, : 3 interactors Detail

More details for 3 interactors identified by 3 citations

Interaction partners 2. rat epithelial Na+ Channel, bP2 region, : 2 : 1 interactors Detail

More details for 3 interactors identified by 1 citations

Experiments performed 24 experiments Detail

1 15N-hsqc

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

2 13C-hsqc, aliphatic and aromatic regions

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

3 HNCO

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

4 HNCaCb

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

5 (Hb)CbCa(CO)NNH

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

6 CCC-TOCSY-NNH

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

7 HCC-TOCSY-NNH

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

8 HCCH-TOCSY (sample 2 only)

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

9 HCCH-COSY (sample 1 only)

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

10 (Hb)CbCa(CO) N CaHa (sample 2 only)

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

11 HaCa N (sample 2 only)

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

12 15N/13C-edited NOESY-HSQC, 50 ms and 150 ms

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

13 HNHa-J

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

14 (Hb)Cb(CgCd)Hd

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

15 (Hb)Cb(CgCd)Ce)He

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

16 HMBC (sample 1 only)

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

17 HNCOCa COHaCa cross (sample 2 only)

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

18 HNCOCa HNHa cross (sample 2 only)

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

19 HaCaHb-J (sample 2 only)

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

20 HN(CO)Hb (sample 1 only)

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

21 HNHb (sample 1 only)

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

22 13C F1-filtered, F3-edited NOESY-HSQC, 250 ms

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

23 long range Jcc (sample 1 only)

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

24 HNCO-trosy (sample 1 only)

Instrument

#1 Model Varian Inova (500 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#2 Model Varian Inova (600 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

#3 Model Varian Inova (800 MHz) Details equipped with pulsed field gradients and triple resonance probes with actively shielded z gradients.

Sample #1

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
1	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
2	rat epithelial Na+ Channel, bP2 region		3.0 mM
3	sodium phosphate		10 mM
4	EDTA		0.005 mM
5	aprotinin		0.01 mM
6	leupeptin		0.005 mM
7	AEBSF		0.005 mM
8	D2O		10 %

Sample #2

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
9	rat Nedd4 WWIII domain	[U-13C; U-15N]	1.0 mM
10	rat epithelial Na+ Channel, bP2 region		3.0 mM
11	sodium phosphate		10 mM
12	EDTA		0.005 mM
13	aprotinin		0.01 mM
14	leupeptin		0.005 mM
15	AEBSF		0.005 mM
16	D2O		100 %

Sample #3

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
17	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
18	rat Nedd4 WWIII domain		3.0 mM
19	sodium phosphate		10 mM
20	EDTA		0.005 mM
21	aprotinin		0.01 mM
22	leupeptin		0.005 mM
23	AEBSF		0.005 mM
24	D2O		10 %

Sample #4

Pressure 1.01325 atm, Temperature 303 (±1) K, pH 6.5 (±0.1)

#	Name	Isotope labeling	Concentration
25	rat epithelial Na+ Channel, bP2 region	[U-13C; U-15N]	1.0 mM
26	rat Nedd4 WWIII domain		3.0 mM
27	sodium phosphate		10 mM
28	EDTA		0.005 mM
29	aprotinin		0.01 mM
30	leupeptin		0.005 mM
31	AEBSF		0.005 mM
32	D2O		100 %

Chemical shift validation 5 contents Detail

Properties

Input source #1: Assigned chemical shifts - Assigned chemical shifts	bmr4963_3.str
Diamagnetism of the molecular assembly	True (excluding Oxygen atoms)
Whether the assembly has a disulfide bond	None
Whether the assembly has a other bond	None
Whether the assembly contains a cyclic polymer	None
Overall data processing status	Warning

Disulfide bonds

None

Other bonds (neither disulfide, covalent nor hydrogen bonds, e.g. Zinc–sulphur bond)

None

Non-standard residues

None

Content subtype: bmr4963_3.str

#	Content subtype	Saveframe	Status	# of rows	Sequence coverage (%)
1	Assigned chemical shifts	shift_set_for_molecule_W	Warning	613	98.0 (chain: 1, length: 50)
2	Assigned chemical shifts	shift_set_for_molecule_B	Warning	191	100.0 (chain: 2, length: 17)
1	Chemical shift references	chemical_shift_reference	Warning	3	No information

Assigned chemical shifts

Saveframe: shift_set_for_molecule_W

Status: Warning

Sequence coverage of experimental data

Entity_assembly_ID: 1, Chain length: 50, Sequence coverage: 98.0%

--------10--------20--------30--------40--------50
GSPVDSNDLGPLPPGWEERTHTDGRVFFINHNIKKTQWEDPRMQNVAITG
| ||||||||||||||||||||||||||||||||||||||||||||||||
G.PVDSNDLGPLPPGWEERTHTDGRVFFINHNIKKTQWEDPRMQNVAITG

Total number of assignments
- ¹³C chemical shifts: 215
- ¹H chemical shifts: 339
- ¹⁵N chemical shifts: 59

Completeness of assignments

Entity_assemble_ID: 1

Excluded Atom_ID in statistics

Comp_index_ID	Comp_ID	Atom_ID	CS value (ppm)
3	PRO	N	144.84
7	ASN	CG	176.99
21	HIS	NE2	169.48
21	HIS	ND1	243.67
30	ASN	CG	176.28
31	HIS	NE2	167.55
31	HIS	ND1	240.06
32	ASN	CG	176.1
37	GLN	CD	180.55
42	ARG	CZ	157.16
42	ARG	NH2	73.1
42	ARG	HH21	6.98
44	GLN	CD	180.05
45	ASN	CG	176.92

All atoms

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹³C chemical shifts	223	208	93.3
¹H chemical shifts	305	294	96.4
¹⁵N chemical shifts	56	53	94.6

Backbone atoms

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹³C chemical shifts	100	92	92.0
¹H chemical shifts	100	97	97.0
¹⁵N chemical shifts	45	42	93.3

Side chain atoms

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹³C chemical shifts	123	116	94.3
¹H chemical shifts	205	197	96.1
¹⁵N chemical shifts	11	11	100.0

Methyl group atoms

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹³C chemical shifts	22	22	100.0
¹H chemical shifts	22	22	100.0

Aromatic group atoms

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹³C chemical shifts	24	18	75.0
¹H chemical shifts	26	20	76.9
¹⁵N chemical shifts	2	2	100.0

Peptide bond of PRO
Tautomeric state of HIS
Rotameric state of ILE , LEU, and VAL
Histogram of normalized assigned chemical shifts
Random coil index and derived parameters (S² and NMR RMSD)
- Chain_ID: 1

Saveframe: shift_set_for_molecule_B
- Status: Warning
- Sequence coverage of experimental data
  - Entity_assembly_ID: 2, Chain length: 17, Sequence coverage: 100.0%
- Total number of assignments
  - ¹³C chemical shifts: 66
  - ¹H chemical shifts: 108
  - ¹⁵N chemical shifts: 17
- Completeness of assignments
  - Entity_assemble_ID: 2
- Peptide bond of PRO
- Rotameric state of ILE , LEU, and VAL
- Histogram of normalized assigned chemical shifts
- Random coil index and derived parameters (S² and NMR RMSD)
  - Chain_ID: 2

Keywords WW domains, PY Motif, Nedd4, ENaC, bP2 peptide, proline-rich

Comp_index_ID	Comp_ID	Atom_ID	CS value (ppm)
5	PRO	N	136.28
7	PRO	N	140.5
10	PRO	N	139.57
11	PRO	N	134.98
12	PRO	N	133.83
13	ASN	CG	176.17

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹³C chemical shifts	74	65	87.8
¹H chemical shifts	96	92	95.8
¹⁵N chemical shifts	13	12	92.3

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹³C chemical shifts	34	27	79.4
¹H chemical shifts	31	29	93.5
¹⁵N chemical shifts	12	11	91.7

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹³C chemical shifts	40	38	95.0
¹H chemical shifts	65	63	96.9
¹⁵N chemical shifts	1	1	100.0

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Found 1 Document (1.48 seconds)

BMRB: 4963

Sample #1

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Related entities 1. rat Nedd4 WWIII domain, : 120 entities Detail

Related entities 2. rat epithelial Na+ Channel, bP2 region, : 1 : 9 : 6 entities Detail

Interaction partners 1. rat Nedd4 WWIII domain, : 3 interactors Detail

Interaction partners 2. rat epithelial Na+ Channel, bP2 region, : 2 : 1 interactors Detail

Experiments performed 24 experiments Detail

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