The muscle-relaxing C-terminal peptide from troponin I populates a nascent helix, facilitating binding to tropomyosin with a potent therapeutic effect.

The conserved C-terminal end segment of troponin I (TnI) plays a critical role in regulating muscle relaxation. This function is retained in the isolated C-terminal 27 amino acid peptide (residues 184-210) of human cardiac TnI (HcTnI-C27): When added to skinned muscle fibers, HcTnI-C27 reduces the Ca2+-sensitivity of activated myofibrils and facilitates relaxation without decreasing the maximum force production. However, the underlying mechanism of HcTnI-C27 function is unknown. We studied the conformational preferences of HcTnI-C27 and a myopathic mutant, Arg192His, (HcTnI-C27-H). Both peptides were mainly disordered in aqueous solution with a nascent helix involving residues from Trp191 to Ile195, as shown by NMR analysis and molecular dynamics simulations. The population of nascent helix was smaller in HcTnI-C27-H than in HcTnI-C27, as shown by circular dichroism (CD) titrations. Fluorescence and isothermal titration calorimetry (ITC) showed that both peptides bound tropomyosin (αTm), with a detectably higher affinity (∼10 µM) of HcTnI-C27 than that of HcTnI-C27-H (∼15 µM), consistent with an impaired Ca2+-desensitization effect of the mutant peptide on skinned muscle strips. Upon binding to αTm, HcTnI-C27 acquired a weakly stable helix-like conformation involving residues near Trp191, as shown by transferred nuclear Overhauser effect spectroscopy and hydrogen/deuterium exchange experiments. With the potent Ca2+-desensitization effect of HcTnI-C27 on skinned cardiac muscle from a mouse model of hypertrophic cardiomyopathy, the data support that the C-terminal end domain of TnI can function as an isolated peptide with the intrinsic capacity of binding tropomyosin, providing a promising therapeutic approach to selectively improve diastolic function of the heart.

The isolated armadillo-repeat domain of Plakophilin 1 is a monomer in solution with a low conformational stability.

Plakophilin 1 (PKP1) is a member of the armadillo repeat family of proteins. It serves as a scaffold component of desmosomes, which are key structural components for cell-cell adhesion. We have embarked on the biophysical and conformational characterization of the ARM domain of PKP1 (ARM-PKP1) in solution by using several spectroscopic (namely, fluorescence and circular dichroism (CD)) and biophysical techniques (namely, analytical ultracentrifugation (AUC), dynamic light scattering (DLS) and differential scanning calorimetry (DSC)). ARM-PKP1 was a monomer in solution at physiological pH, with a low conformational stability, as concluded from DSC experiments and thermal denaturations followed by fluorescence and CD. The presence or absence of disulphide bridges did not affect its low stability. The protein unfolded through an intermediate which has lost native-like secondary structure. ARM-PKP1 acquired a native-like structure in a narrow pH range (between pH 6.0 and 8.0), indicating that its adherent properties might only work in a very narrow pH range.

Competitive inhibition of protein adsorption to silica surfaces by their coating with high density charge polyelectrolytes.

The adsorption of proteins to silica surface is a common process mainly governed by the electrostatic attractive interaction between the pH-dependent negatively silica surface and the positive charges of the biomolecule. This process often reduces the conformational stability of the adsorbed protein and may reduce its biological functionality mostly due to multimolecular processes such as aggregation and fibrillation. Here we show that high-density charge cationic polyelectrolytes may successfully compete with the protein for the silica surface containing deprotonated-silanol groups. Therefore, the coating of silica surfaces with these cationic polyelectrolytes precludes the adsorption of the protein to the solid surface. Intensive water washing of the polyelectrolyte-coated silica surfaces had does not result in polyelectrolyte release (even at moderate ionic strength) maintaining the solid surface protected from protein adsorption.

Human importin α3 and its N-terminal truncated form, without the importin-ß-binding domain, are oligomeric species with a low conformational stability in solution.

BACKGROUND: Eukaryotic cells have a continuous transit of macromolecules between the cytoplasm and the nucleus. Several carrier proteins are involved in this transport. One of them is importin α, which must form a complex with importin ß to accomplish its function, by domain-swapping its 60-residue-long N terminus. There are several human isoforms of importin α; among them, importin α3 has a particularly high flexibility. METHODS: We studied the conformational stability of intact importin α3 (Impα3) and its truncated form, where the 64-residue-long, N-terminal importin-ß-binding domain (IBB) has been removed (ΔImpα3), in a wide pH range, with several spectroscopic, biophysical, biochemical methods and with molecular dynamics (MD). RESULTS: Both species acquired native-like structure between pH 7 and 10.0, where Impα3 was a dimer (with an apparent self-association constant of ~10 µM) and ΔImpα3 had a higher tendency to self-associate than the intact species. The acquisition of secondary, tertiary and quaternary structure, and the burial of hydrophobic patches, occurred concomitantly. Both proteins unfolded irreversibly at physiological pH, by using either temperature or chemical denaturants, through several partially folded intermediates. The MD simulations support the presence of these intermediates. CONCLUSIONS: The thermal stability of Impα3 at physiological pH was very low, but was higher than that of ΔImpα3. Both proteins were stable in a narrow pH range, and they unfolded at physiological pH populating several intermediate species. GENERAL SIGNIFICANCE: The low conformational stability explains the flexibility of Impα3, which is needed to carry out its recognition of complex cargo sequences.

The C Terminus of the Ribosomal-Associated Protein LrtA Is an Intrinsically Disordered Oligomer.

The 191-residue-long LrtA protein of Synechocystis sp. PCC 6803 is involved in post-stress survival and in stabilizing 70S ribosomal particles. It belongs to the hibernating promoting factor (HPF) family, intervening in protein synthesis. The protein consists of two domains: The N-terminal region (N-LrtA, residues 1⁻101), which is common to all the members of the HPF, and seems to be well-folded; and the C-terminal region (C-LrtA, residues 102⁻191), which is hypothesized to be disordered. In this work, we studied the conformational preferences of isolated C-LrtA in solution. The protein was disordered, as shown by computational modelling, 1D-¹H NMR, steady-state far-UV circular dichroism (CD) and chemical and thermal denaturations followed by fluorescence and far-UV CD. Moreover, at physiological conditions, as indicated by several biochemical and hydrodynamic techniques, isolated C-LrtA intervened in a self-association equilibrium, involving several oligomerization reactions. Thus, C-LrtA was an oligomeric disordered protein.

The histidine phosphocarrier protein, HPr, binds to the highly thermostable regulator of sigma D protein, Rsd, and its isolated helical fragments.

The phosphotransferase system (PTS) controls the preferential use of sugars in bacteria and it is also involved in other processes, such as chemotaxis. It is formed by a protein cascade in which the first two proteins are general (namely, EI and HPr) and the others are sugar-specific permeases. The Rsd protein binds specifically to the RNA polymerase (RNAP) σ70 factor. We first characterized the conformational stability of Escherichia coli Rsd. And second, we delineated the binding regions of Streptomyces coelicolor, HPrsc, and E. coli Rsd, by using fragments derived from each protein. To that end, we used several biophysical probes, namely, fluorescence, CD, NMR, ITC and BLI. Rsd had a free energy of unfolding of 15 kcal mol-1 at 25 °C, and a thermal denaturation midpoint of 103 °C at pH 6.5. The affinity between Rsd and HPrsc was 2 µM. Interestingly enough, the isolated helical-peptides, comprising the third (RsdH3) and fourth (RsdH4) Rsd helices, also interacted with HPrsc in a specific manner, and with affinities similar to that of the whole Rsd. Moreover, the isolated peptide of HPrsc, HPr9-30, comprising the active site, His15, also was bound to intact Rsd with similar affinity. Therefore, binding between Rsd and HPrsc was modulated by the two helices H3 and H4 of Rsd, and the regions around the active site of HPrsc. This implies that specific fragments of Rsd and HPrsc can be used to interfere with other protein-protein interactions (PPIs) of each other protein.

The Monomeric Species of the Regulatory Domain of Tyrosine Hydroxylase Has a Low Conformational Stability.

Tyrosine hydroxylase (TyrH) catalyzes the hydroxylation of tyrosine to form 3,4-dihydroxyphenylalanine, the first step in the synthesis of catecholamine neurotransmitters. The protein contains a 159-residue regulatory domain (RD) at its N-terminus that forms dimers in solution; the N-terminal region of RDTyrH (residues 1-71) is absent in the solution structure of the domain. We have characterized the conformational stability of two species of RDTyrH (one containing the N-terminal region and another lacking the first 64 residues) to clarify how that N-terminal region modulates the conformational stability of RD. Under the conditions used in this study, the RD species lacking the first 64 residues is a monomer at pH 7.0, with a small conformational stability at 25 °C (4.7 ± 0.8 kcal mol(-1)). On the other hand, the entire RDTyrH is dimeric at physiological pH, with an estimated dissociation constant of 1.6 µM, as determined by zonal gel filtration chromatography; dimer dissociation was spectroscopically silent to circular dichroism but not to fluoresecence. Both RD species were disordered below physiological pH, but the acquisition of secondary native-like structure occurs at pHs lower than those measured for the attainment of tertiary native- and compactness-like arrangements.