Table 3.

Hairpin stability effects of mutations and truncations in the HP7 hairpin series. Measurements are at pH 6 unless otherwise noted.

Name	Sequence	Tm (	°C)	%-folded	at 298K
		CDa	NMRb	strandc	turnd
HP7	KTW-NPATGK-WTE	66 (63)	69	88	89 ± 1
	+ 8% HFIP, pH ≈5.5	59 (54)	57	85	82 ± 1
	at pH 3		47	77	79 ± 2
Strand Mutations and Truncations
K1R	RTW-NPATGK-WTE	71 (68)	71	90	<90>
K1A/E12A	ATW-NPATGK-WTA	43 (43)	48	74	82 ± 2
HP7(-2)	Ac-TW-NPATGK-WT-NH2	22 (22)	17	(41b)	55 ± 6
HP7(-4)	Ac-W-NPATGK-W-NH2	~1e	< 7e	(24b)	41 ± 9
Des-W10	KTW-NPATGK-TE	n. a.	n. a.	n. a.	20 ± 10
loop	Ac-NPATGK-NH2	n. a.	n. a.	n. a.	6 ± 7
Loop Mutations
N4D	KTW-DPATGK-WTE	66 (62)	73	87	89 ± 1
N4A	KTW-APATGK-WTE	−1 (≤15)	13 f	41	43 ± 2
P5A	KTW-NAATGK-WTE	61 (59)	56	88	81 ± 7
T7A	KTW-NPAAGK-WTE	57 (55)	65	83	80 ± 15
G8A	KTW-NPATAK-WTE	45 (44)	56	81	n. a.
Substitution with Shorter Loops
INGK	KTW- INGK -WTE	54 (49)	42	63(67b)	(63 – 70)
NPDGT	KTW- NPDGT -WTE	32g(50)	< 7g	39(29b)	(46 / 50)
EPDGK	KTW- EPDGK -WTE	≪ 5	≪ 7	23(15b)	(23 / 31 )

^aThe CD Tm is derived assuming a warming-induced decrease (0.36%/ °C, see Methods) in the folding-induced change in [θ]₂₂₈ with Δ[θ]₂₂₈ = +434,000° at 0 °C for all systems. The model-free Tm’s based on the second derivative of the fitted melting curve are shown in parentheses; italicized values are viewed as inaccurate due to the near linearity of the CD melt.

^bBased on the W3-Hε3 shift.

^cBased on T2 and W10 Hα and T2, W3, and T11 H_N CSDs.

^dFor the 6-residue loop species, the average and standard deviation of %-folded as determined from the T7 and K9 H_N CSDs and G8-ΔδHα, assuming that (K1R)-HP7 is 90% folded at 298K. See text for the probes employed for the 5-residue loops and the INGK turn.

^e38% folded at 7 °C, 24% at 25 °C based on the Hε3 shift probe versus 48% folded at 5 °C based on CD.

^fTwo additional NMR melting points are available: 12 °C (strand-CSDs) and 17 °C (turn CSDs).

^g34% folded at 280K based on the W3-Hε3 shift; 61% by CD assuming the expectation values for HP7 applies to this loop.