Structural basis for unfolding pathway-dependent stability of proteins: vectorial unfolding versus global unfolding

Abstract

Point mutations in proteins can have different effects on protein stability depending on the mechanism of unfolding. In the most interesting case of I27, the Ig-like module of the muscle protein titin, one point mutation (Y9P) yields opposite effects on protein stability during denaturant-induced "global unfolding" versus "vectorial unfolding" by mechanical pulling force or cellular unfolding systems. Here, we assessed the reason for the different effects of the Y9P mutation of I27 on the overall molecular stability and N-terminal unraveling by NMR. We found that the Y9P mutation causes a conformational change that is transmitted through beta-sheet structures to reach the central hydrophobic core in the interior and alters its accessibility to bulk solvent, which leads to destabilization of the hydrophobic core. On the other hand, the Y9P mutation causes a bend in the backbone structure, which leads to the formation of a more stable N-terminal structure probably through enhanced hydrophobic interactions.

Figure 1

Structure of I27 and import of its Y9 mutants. (A) Cartoon diagram showing the β-sandwich structure of the I27 molecule (PDB ID, 1TIT). Tyr9 is shown in stick form. (B) Effects of mutations at residue 9 of the I27 domain on the import of radiolabeled pb₂(80)-I27 fusion proteins. Mitochondria were isolated from yeast strain D273-10B. The indicated radiolabeled proteins were incubated with mitochondria at 25°C for indicated times in the absence (+ΔΨ) or presence (−ΔΨ) of valinomycin, which dissipates the membrane potential (ΔΨ) across the inner membrane. The mitochondria were treated with (+Proteinase K) or without (−Proteinase K) proteinase K, and radioactive proteins were analyzed by SDS-PAGE and radioimaging (left panels). The amounts of radiolabeled proteins added to each reaction were set to 100% and imported proteins were plotted against incubation times (right panels). 10%, ten percent of the radiolabled proteins added to each reaction. (C) Imported, proteinase-K-protected fractions in (B) were quantified, and import rates (initial slopes of the import reactions) were plotted. Values are presented as mean ± SD.

Figure 2

CD and NMR spectra and H-D exchange of I27 and Y9 mutants. (A) CD spectra of the I27 mutants. Far-UV CD spectra of 10 μM I27 mutants were recorded in 50 mM KPi (pH 7.4) at 25°C. (B) Chemical shift changes of backbone amides of I27 derivatives as compared with wild-type (WT) I27. Chemical shift changes were calculated in [¹H, ¹⁵N]-HSQC spectra according to the equation, [Δδ(¹H)² + (Δδ(¹⁵N)/7)²]^1/2. (C) Residues of the I27 derivatives were colored in ribbon form according to chemical shift changes as compared with WT I27: >0.50 ppm (red), 0.25–0.50 ppm (orange), 0.05–0.25 ppm (yellow) and 0-0.05 ppm (blue).

Figure 3

Secondary structures and H-D exchange of I27 and Y9P. (A) Upper panel: secondary structure diagrams of WT I27 and Y9P (estimated from the NMR structure [Table II] by PROCHECK). PDB ID codes for each structure are shown. Central and lower panels: NMR chemical shift indexes for WT and Y9P (this study) reflecting secondary structures. (B) Slowly exchanging NH signals for WT I27 (upper panel) and Y9P I27 (lower panel) observed at 300 min after H-D exchange. Note that the NH signal of V11 is observed only for WT I27, but not for Y9P. (C) PFs for H-D exchange of backbone NHs in WT and Y9P I27. Bars reaching logPF = 6, exchange too fast (log PF ≥ 6); bars with log PF = 1, exchange too slow (log PF ≤ 1). P, Pro residue. (D) PF (protection factor) values of H-D exchange of backbone NHs of each residue are mapped onto the molecular structure of I27. Protection factors are calculated from the equation, PF = k_ex(U)/k_ex(N), where k_ex(N) and k_ex(U) represent the rates of exchange for a given NH under folded and unfolded conditions, respectively. The exchange rates of fully solvent-exposed NHs, k_ex(U), were estimated on the basis of the primary structure, pH, and temperature using a program SPHERE (http://www.fccc.edu/research/labs/roder/sphere). Residues are colored according to PF values; log PF > 5.5 (red), log PF = 2.5–5.5 (yellow), and log PF < 2.5 (blue). Tyr9 is shown in stick form.

Figure 4

Mapping of the residues affected by the Y9P mutation. (A) The residues proposed to form a central hydrophobic core of the I27 domain are shown in stick form (green): F21, I23, W34, H56, L58, V71, and F73. (B) The residues that showed large chemical shift changes (>0.05 ppm) are shown in stick form (yellow): V4, L8, G10, V11, E12, H20, F21, E22, I23, E24, E27, V30, G38, I50, E51, I57, L58, H61, F73, Q74, K79, S80, A81, A82, and K85. (C) The residues included in (A) and (B) are shown in red: F21, I23, L58, and F73.

Figure 5

Effects of the Y9P mutation on the N-terminal structure of the I27 molecule. (A) Main-chain structures of WT (blue) and Y9P (pink) I27 are superimposed on the basis of secondary structure matching using a program CCP4 suite. RMSD of the two structures is 2.04 Å. (B) Overlay of the 24 (WT) and 20 (Y9P) NMR structures (left panels) and their close-up around residue 9 (right panels). Residue 9 is shown in red and distances between the C_α atoms of L8 and F14 are indicated. (C) The N-terminal structures around residue 9 of WT (blue) and Y9P (pink) I27 are superimposed as in (A). Distances between C_α atoms in the two structures are shown in parentheses. (D) Relative geometry of hydrophobic residues (L8, V11, V13, and F21) in the N-terminal region for the NMR structures of WT I27 and Y9P I27. Distances (Å) between the C atom pairs specified with broken lines are indicated. (E) Comparison of the relative geometry of the β-strand pairs A-B and A′-G between WT I27 and Y9P I27. Distances (Å) for V4C_β–L25C_β, K6C_β–E24C_β, V11C_β– L84C_β, and V13C_β–V86C_β are indicated. β-strands are shown with shaded arrows, and C_α atoms are indicated by dots.

Figure 6

Effects of the Y9P mutation on the central core domain of the I27 molecule. (A) Side chains of the central core residues are shown for WT (blue) and Y9P (pink) I27. Orientations of the side chains of F21 in both structures are adjusted for the best match. (B) The WT (blue) and Y9P (pink) I27 structures are drawn in space-filling form, showing the channels leading to the central hydrophobic cores (F21, I23, W34, H56, L58, V71, and F73; yellow) in the interiors. The residues constituting the channels or cavities leading to the hydrophobic cores are shown in cyan and the volumes of the cavities were calculated by the program CASTp available on the Internet (http://sts-fw.bioengr.uic.edu/castp/index.php). (C) L36, L41, and M67, which form the entrance of the channel in WT I27, are shown in red in the WT and Y9P I27 structures drawn as in (B). (D) The same view of the WT (blue) and Y9P (pink) I27 structures as in (B) are shown in ribbon form. Side chains of the central hydrophobic core residues (yellow) and channel forming residues (L36, L41, and M67; red) are indicated in stick form.