The anisotropic component of the magnetic susceptibility tensor (Δχ tensor) associated with various paramagnetic metal ions can induce pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs) in proteins, yielding valuable restraints in structural studies. In particular, PCSs have successfully been used to study ligands that bind to proteins tagged with a paramagnetic metal ion, which is of great interest in fragment-based drug design. To create easy-to-interpret PCSs, the metal ion must be attached to the protein in a rigid manner. Most of the existing methods for site-specific attachment of a metal tag, however, result in tethers with residual flexibility. Here we present model calculations to quantify the extent, to which mobility of the metal-binding tag can compromise the quality of the Δχ tensor that can be determined from the PCSs observed in the protein. Assuming that the protein can be approximated by a sphere and the tag is attached by a single tether, the results show that a single effective ∆χ tensor can describe the PCSs and RDCs of the protein spins very well even in the presence of substantial tag mobility, implying that PCSs of ligands in binding pockets of the protein can be predicted with similar accuracy. In contrast, the quality of the PCS prediction for nuclear spins positioned above the surface of the protein is significantly poorer, with implications for studies of protein-protein complexes. The simulations probed the sensitivity of the effective Δχ tensor to different parameters, including length of the tether between protein and metal ion, protein size, type and amplitude of tag motion, tensor orientation relative to the protein and direction of tag motion. Tether length and amplitude of motion were identified as two key parameters. It is shown that the amplitude of tag motions cannot be quantified by simple comparisons of the effective Δχ tensor with the alignment tensor determined from RDCs.

Glycolysis and transmembrane cation flux (measured by NMR) are accelerated in red blood cells controllably distorted in gels.

Single-stranded DNA (ssDNA)-binding protein (SSB) protects ssDNA from degradation and recruits other proteins for DNA replication and repair. Escherichia coli SSB is the prototypical eubacterial SSB in a family of tetrameric SSBs. It consists of a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain). The eight-residue C-terminal segment of SSB (C-peptide) mediates the binding of SSB to many different SSB-binding proteins. Previously published nuclear magnetic resonance (NMR) data of the monomeric state at pH 3.4 showed that the C-peptide binds to the OB-domain at a site that overlaps with the ssDNA binding site, but investigating the protein at neutral pH is difficult because of the high molecular mass and limited solubility of the tetramer. Here we show that the C-domain is highly mobile in the SSB tetramer at neutral pH and that binding of the C-peptide to the OB-domain is so weak that most of the C-peptides are unbound even in the absence of ssDNA. We address the problem of determining intramolecular binding affinities in the situation of fast exchange between two states, one of which cannot be observed by NMR and cannot be fully populated. The results were confirmed by electron paramagnetic resonance spectroscopy and microscale thermophoresis. The C-peptide-OB-domain interaction is shown to be driven primarily by electrostatic interactions, so that binding of 1 equiv of (dT)35 releases practically all C-peptides from the OB-domain tetramer. The interaction is much more sensitive to NaCl than to potassium glutamate, which is the usual osmolyte in E. coli. As the C-peptide is predominantly in the unbound state irrespective of the presence of ssDNA, long-range electrostatic effects from the C-peptide may contribute more to regulating the activity of SSB than any engagement of the C-peptide by the OB-domain.

Single-stranded DNA (ssDNA) binding protein (SSB) is an essential protein to protect ssDNA and recruit specific ssDNA-processing proteins. Escherichia coli SSB forms a tetramer at neutral pH, comprising a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain) of ∼64 amino acid residues. The C-terminal eight-residue segment of SSB (C-peptide) has been shown to interact with the OB-domain, but crystal structures failed to reveal any electron density of the C-peptide. Here we show that SSB forms a monomer at pH 3.4, which is suitable for studies by high-resolution nuclear magnetic resonance (NMR) spectroscopy. The OB-domain retains its 3D structure in the monomer, and the C-peptide is shown by nuclear Overhauser effects and lanthanide-induced pseudocontact shifts to bind to the OB-domain at a site that harbors ssDNA in the crystal structure of the SSB–ssDNA complex. 15N relaxation data demonstrate high flexibility of the polypeptide segment linking the C-peptide to the OB-domain and somewhat increased flexibility of the C-peptide compared with the OB-domain, suggesting that the C-peptide either retains high mobility in the bound state or is in a fast equilibrium with an unbound state.

Excitation-contraction coupling (ECC) is a physiological process that links excitation of muscles by the nervous system to their mechanical contraction. In skeletal muscle, ECC is initiated with an action potential, generated by the somatic nervous system, which causes a depolarisation of the muscle fibre membrane (sarcolemma). This leads to a rapid change in the transmembrane potential, which is detected by the voltage-gated Ca 2+ channel dihydropyridine receptor (DHPR) embedded in the sarcolemma. DHPR transmits the contractile signal to another Ca 2+ channel, ryanodine receptor (RyR1), embedded in the membrane of the sarcoplasmic reticulum (SR), which releases a large amount of Ca 2+ ions from the SR that initiate muscle contraction. Despite the fundamental role of ECC in skeletal muscle function of all vertebrate species, the molecular mechanism underpinning the communication between the two key proteins involved in the process (DHPR and RyR1) is still largely unknown. The goal of this work is to review the recent progress in our understanding of ECC in skeletal muscle from the point of view of the structure and interactions of proteins involved in the process, and to highlight the unanswered questions in the field.