Titin PEVK segment: charge-driven elasticity of the open and flexible polyampholyte

Abstract

The giant protein titin spans half of the sarcomere length and anchors the myosin thick filament to the Z-line of skeletal and cardiac muscles. The passive elasticity of muscle at a physiological range of stretch arises primarily from the extension of the PEVK segment, which is a polyampholyte with dense and alternating-charged clusters. Force spectroscopy studies of a 51 kDa fragment of the human fetal titin PEVK domain (TP1) revealed that when charge interactions were reduced by raising the ionic strength from 35 to 560 mM, its mean persistence length increased from 0.30 +/- 0.04 nm to 0.60 +/- 0.07 nm. In contrast, when the secondary structure of TP1 was altered drastically by the presence of 40 and 80% (v/v) of trifluoroethanol, its force-extension behavior showed no significant shift in the mean persistence length of approximately approximately 0.18 +/- 0.03 nm at the ionic strength of 15 mM. Additionally, the mean persistence length also increased from 0.29 to 0.41 nm with increasing calcium concentration from pCa 5-8 to pCa 3-4. We propose that PEVK is not a simple entropic spring as is commonly assumed, but a highly evolved, gel-like enthalpic spring with its elasticity dominated by the sequence-specific charge interactions. A single polyampholyte chain may be fine-tuned to generate a broad range of molecular elasticity by varying charge pairing schemes and chain configurations.

Fig. 1

Charge profiles of human PEVK exon modules. PEVK module sequences were deduced from exon DNA sequences (AJ277892, 11/ 23/2001) by including the final G in the preceding exon as the first base. A total of 113 PEVK exons were classified into two groups: (a) 100 PEVK exons designated as Group P, (b) 13 PEVK modules designated as Group E. (a, b) The tables include the module (exon) number and its corresponding sequence. The corresponding module size (number of amino acids), pI and the module composition of human soleus (designated as s’), fetal (designated as f’) and cardiac (designated as c’) PEVK of titin isoforms are listed to the right of the table. All acidic amino acids (e and d) and basic amino acids (K, R, H) were marked with red and blue backgrounds, respectively. The charge profile for most of modules in Group P is shown on the bottom of the table (a), with +, −, × indicating basic (blue), acidic (red) and other amino acids, respectively. (c) The sarcomere location of the PEVK segment in titin that spans from the Z-line to the M-line. The sequence of 18 PEVK modules that make up the TP1 fragment of the human fetal skeletal titin PEVK segment (AF321609) are shown (d), with the exon numbering scheme used by Bang et al. (2001) and the acidic and basic residues highlighted in red and blue respectively. The charge profile along TP1 at pH 7.0 with a 5-residue window showing an exceptionally high density of alternating charges in TP1 were calculated using the ISOELECTRIC program from the GCG Wisconsin Package. Our TP1 sequence differs from that reported by Bang et al. (2001) as follows: T, K, L and I, instead of A, QR, V, V in exons 167, 169, 174 and 177 (marked in yellow). The residues in lower case italic (in green) were extras added during cloning. Further details of TP1 may be found elsewhere (Gutierrez-Cruz et al., 2001). (e) Charges along cardiac PEVK sequence calculated as for TP1. Cardiac PEVK is assembled from seven different modules and its sequence is shown in the inset (Bang et al., 2001).

Fig. 2

WLC modeling of TP1 elasticity and distribution of elastic parameters. (a) A representative force-extension curve showing the curve fitting with WLC parameters of persistence length P=0.14 nm, detachment force Df=470 pN, and segment length L=110 nm. See (Wang et al., 2001) for details of different models of polymer elasticity. The zero point for data fitting is assumed to be the Z position of the retraction curve where the force is equivalent to that at the non-interacting equilibrium position. The inset is a plot of F^−½ vs. z, which linearizes the WLC force response at large extensions. (b) Scatter plot of persistence length as a function of segment length of 61 force-distance curves (TP1, ionic strength=140 mM, pH=7.0, pCa=7.0 at 23°C). See (Forbes et al., 2001) for details of experimental setup.

Fig. 3

Effects of ionic strength and TFE on TP1 elasticity. (a) The mean persistence lengths (closed circles with standard error bars, ●) and the Debye–Hückel screening length (inverted triangles, ▽) as a function of ionic strength from 35 to 560 mM. (b) The segment lengths (closed triangles, ▲) and detachment forces (open circles, ○) as a function of ionic strength. Error bars denote one standard deviation computed from the distributions. Experiments were carried out at room temperature (23°C) in buffer B (10 mM imidazole, 10 mM K-EGTA, 2.1 mM CaCl₂, 2.5 mM MgCl₂, 2 mM NaN₃, pH 7.0, pCa 7.0), with the ionic strength adjusted with KCl. Free [Mg²⁺]=2.0 mM. pCa=7.0±0.1. (c) The mean persistence lengths (closed circles with standard error bars, ●) as a function of TFE concentration. (d) The segment lengths and detachment forces as a function of TFE concentration. Error bars denote one standard deviation computed from the distributions. Data were taken at 2°C in 10 mM potassium phosphate buffer (pH=7 and ionic strength at 15 mM). Data collection and analysis as in Figure 2.

Fig. 4

CD spectra for TP1 at different ionic strengths, temperatures and TFE concentrations. (a) CD spectra at ionic strengths of 0, 35, 70, 140, 280, and 560 mM KCl in 10 mM MOPS, pH7.0 with 10 mM K-EGTA, 2.1 mM CaCl₂, 2.5 mM MgCl₂, 2 mM NaN₃, at 23°C. (b) CD spectra at ionic strengths of 0 mM, 35, 70, 140, 280 mM, and 560 mM KCl in 60% TFE in 10 mM potassium phosphate, pH 7.0, at 23°C. (c, d) CD spectra at 2 and 23°C at 0, 40 and 80% TFE in 10 mM potassium phosphate, pH 7.0. Inset: difference CD spectra. Concentration of TP1 was 5 μM for all experiments. Spectra were recorded with a 1.0 nm bandwidth and resolution of 0.1 nm over the wavelength range 190 to 250 nm in a 0.1 cm path length cell. See (Ma and Wang, 2002) for further experimental details.

Fig. 5

Molecular dynamics simulations demonstrating the manifold of configurations that PEVK can adopt after synthesis and stretching. (a) Probable structures for the fetal PEVK exons 170–173 (Gutierrez-Cruz et al., 2001) were generated using the TRADES software package (Feldman and Hogue, 2000). Plausible conformers were generated with FOLDTRAJ. Six with the smallest radius of gyration and one with the largest were selected and put into a water box such that at least 5Å of water surrounded the protein, with five Cl⁻ ions to neutralize the system. The systems were minimized for 1000 steps and subjected to 1000 ps of molecular dynamics simulation using NAMD (Kale et al., 1999) with a molecular interaction cutoff of 10Å. The simulations were setup and visualized with VMD (Humphrey et al., 1996). Selected lysine and glutamate residues are highlighted showing how neighboring chains interact through the charge pairs. The oxygen atoms on the glutamate residues are shown in red and the nitrogen atom on the lysine residues in blue. Color coded numbers indicate the residues forming salt bridges. The structures at the top are three of many possible configurations that the polypeptide can adopt. When the polypeptide is stretched, the chain becomes extended and the charge groups can no longer interact. The middle panel shows a highly extended (although not fully) configuration with all of the charge groups highlighted. Upon release, compact configurations reform with different charge pairing schemes as shown in the bottom panel. In each structure the N’ indicates the amino terminus of the polypeptide chain. This study utilized the high-performance computational capabilities of the Biowulf PC/Linux cluster at the NIH, Bethesda, MD (http://biowulf.nih.gov). (b) One polyampholyte-many springs. Two different wrapping’ schemes of charge interactions may lead to distinct persistence lengths. The mechanical stretching extends polypeptides over length scales of tens to hundreds of nanometers, and affects charge interactions at the nanometer scale that is comparable to the charge screening length, λ.