Molecular Characterisation of Titin N2A and Its Binding of CARP Reveals a Titin/Actin Cross-linking Mechanism

Striated muscle responds to mechanical overload by rapidly up-regulating the expression of the cardiac ankyrin repeat protein, CARP, which then targets the sarcomere by binding to titin N2A in the I-band region. To date, the role of this interaction in the stress response of muscle remains poorly understood. Here, we characterise the molecular structure of the CARP-receptor site in titin (UN2A) and its binding of CARP. We find that titin UN2A contains a central three-helix bundle fold (ca 45 residues in length) that is joined to N- and C-terminal flanking immunoglobulin domains by long, flexible linkers with partial helical content. CARP binds titin by engaging an α-hairpin in the three-helix fold of UN2A, the C-terminal linker sequence, and the BC loop in Ig81, which jointly form a broad binding interface. Mutagenesis showed that the CARP/N2A association withstands sequence variations in titin N2A and we use this information to evaluate 85 human single nucleotide variants. In addition, actin co-sedimentation, co-transfection in C2C12 cells, proteomics on heart lysates, and the mechanical response of CARP-soaked myofibrils imply that CARP induces the cross-linking of titin and actin myofilaments, thereby increasing myofibril stiffness. We conclude that CARP acts as a regulator of force output in the sarcomere that preserves muscle mechanical performance upon overload stress

Linkage between fitness of yeast cells and adenylate kinase catalysis

Enzymes have evolved with highly specific values of their catalytic parameters kcat and KM. This poses fundamental biological questions about the selection pressures responsible for evolutionary tuning of these parameters. Here we are address these questions for the enzyme adenylate kinase (Adk) in eukaryotic yeast cells. A plasmid shuffling system was developed to allow quantification of relative fitness (calculated from growth rates) of yeast in response to perturbations of Adk activity introduced through mutations. Biophysical characterization verified that all variants studied were properly folded and that the mutations did not cause any substantial differences to thermal stability. We found that cytosolic Adk is essential for yeast viability in our strain background and that viability could not be restored with a catalytically dead, although properly folded Adk variant. There exist a massive overcapacity of Adk catalytic activity and only 12% of the wild type kcat is required for optimal growth at the stress condition 20°C. In summary, the approach developed here has provided new insights into the evolutionary tuning of kcat for Adk in a eukaryotic organism. The developed methodology may also become useful for uncovering new aspects of active site dynamics and also in enzyme design since a large library of enzyme variants can be screened rapidly by identifying viable colonies

Choosing the optimal spectroscopic toolkit to understand protein function

Spectroscopy was one of the earliest methods used to study the properties and reactions of proteins, and remains one of the most powerful and widely used approaches to this day. A sometimes bewildering range of spectroscopies is now available, applicable to different sample states, timescales and indeed biological questions. This editorial describes some of the most relevant spectroscopic methods together with a selection of illustrative examples.</jats:p

New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein

A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a beta-sheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF)-alpha. The bioactive form of this validated pharmacological target protein is a non-covalently linked homo-trimer. This structural feature leads to the observation of a certain heterogeneity concerning the binding mode of TNF-alpha binding molecules, for instance in terms of monomer/trimer specificity. We analyzed a ubiquitin-based TNF-alpha binder, selected by ribosome display, with a particular focus on its mode of interaction. Using enzyme-linked immunosorbent assays, specific binding to TNF-alpha with nanomolar affinity was observed. In isothermal titration calorimetry we obtained comparable results regarding the affinity and detected an exothermic reaction with one ubiquitin-derived binding molecule binding one TNF-alpha trimer. Using NMR spectroscopy and other analytical methods the 1∶3 stoichiometry could be confirmed. Detailed binding analysis showed that the interaction is affected by the detergent Tween-20. Previously, this phenomenon was reported only for one other type of alternative scaffold-derived binding proteins – designed ankyrin repeat proteins – without further investigation. As demonstrated by size exclusion chromatography and NMR spectroscopy, the presence of the detergent increases the association rate significantly. Since the special architecture of TNF-alpha is known to be modulated by detergents, the access to the recognized epitope is indicated to be restricted by conformational transitions within the target protein. Our results suggest that the ubiquitin-derived binding protein targets a new epitope on TNF-alpha, which differs from the epitopes recognized by TNF-alpha neutralizing antibodies

Optimisation of Over-Expression in E. coli and Biophysical Characterisation of Human Membrane Protein Synaptogyrin 1

Progress in functional and structural studies of integral membrane proteins (IMPs) is lacking behind their soluble counterparts due to the great challenge in producing stable and homogeneous IMPs. Low natural abundance, toxicity when over-expressed and potential lipid requirements of IMPs are only a few reasons for the limited progress. Here, we describe an optimised workflow for the recombinant over-expression of the human tetraspan vesicle protein (TVP) synaptogyrin in Escherichia coli and its biophysical characterisation. TVPs are ubiquitous and abundant components of vesicles. They are believed to be involved in various aspects of the synaptic vesicle cycle, including vesicle biogenesis, exocytosis and endocytotic recycling. Even though TVPs are found in most cell types, high-resolution structural information for this class of membrane proteins is still missing. The optimisation of the N-terminal sequence of the gene together with the usage of the recently developed Lemo21(DE3) strain which allows the balancing of the translation with the membrane insertion rate led to a 50-fold increased expression rate compared to the classical BL21(DE3) strain. The protein was soluble and stable in a variety of mild detergents and multiple biophysical methods confirmed the folded state of the protein. Crosslinking experiments suggest an oligomeric architecture of at least four subunits. The protein stability is significantly improved in the presence of cholesteryl hemisuccinate as judged by differential light scattering. The approach described here can easily be adapted to other eukaryotic IMPs

All atom insights into the impact of crowded environments on protein stability by NMR spectroscopy

The high density of macromolecules affecting proteins due to volume exclusion has been discussed in theory but numerous in vivo experiments cannot be sufficiently understood taking only pure entropic stabilization into account. Here, we show that the thermodynamic stability of a beta barrel protein increases equally at all atomic levels comparing crowded environments with dilute conditions by applying multidimensional high-resolution NMR spectroscopy in a systematic manner. Different crowding agents evoke a pure stabilization cooperatively and do not disturb the surface or integrity of the protein fold. The here developed methodology provides a solid base that can be easily expanded to incorporate e.g. binding partners to recognize functional consequences of crowded conditions. Our results are relevant to research projects targeting soluble proteins in vivo as it can be anticipated that their thermodynamic stability increase comparably and has consequently to be taken into account to coherently understand intracellular processes.publishe

Insights into protein stability in cell lysate by 19F NMR spectroscopy

In living organisms, protein folding and function take place in an inhomogeneous, highly crowded environment possessing a concentration of diverse macromolecules of up to 400 g/L. It has been shown that the intracellular environment has a pronounced effect on the stability, dynamics and function of the protein under study and has for this reason to be considered. However, most protein studies are neglecting the presence of these macromolecules. Consequently, we probe here the overall thermodynamic stability of cold shock protein B from  Bacillus subtilis  ( Bs CspB) in cell lysate. We found that an increase in cell lysate concentration causes a monotonic increase in thermodynamic stability of  Bs CspB. This result strongly underlines the importance of considering the biological environment when inherent protein parameters shall be quantitatively determined. Moreover, we demonstrate that the targeted application of  19 F NMR spectroscopy operates as an ideal tool utilized to protein studies performed in complex cellular surroundings.publishe

Macromolecular crowding tunes protein stability by manipulating solvent accessibility

In all intracellular processes, protein structure and dynamics are subjected to the influence of macromolecular crowding (MC). Here, the impact of different types and sizes of MC agents on the model protein BsCspB are comprehensively investigated under thermal as well as chemical denaturation. We consistently reveal a distinct stabilization of BsCspB in dependence on the MC concentration but not on viscosity, polarity or size of MC agent used. This general stabilization is decoded by using NMR spectroscopy via monitoring chemical shift (CS) perturbations, the intramolecular hydrogen bonding network as well as local protection of amide protons against exchange with solvent protons. Whereas CSs and the hydrogen bonding network are not systematically affected in presence of MC, we detected a pronounced reduced exchange in loop regions of BsCspB. We conclude that this reduced accessibility of solvent protons acts as a key parameter for the increase in protein stability seen under MC.publishe

Targeted expression and purification of fluorine labelled cold shock protein B by using an auxotrophic strategy

High resolution NMR spectroscopy is a seminal method in modern structural biology to obtain insights into proteins' structure, dynamics and function at dilute condition as well as in a cell-like environment or even intracellularly. Usually, 1H, 15N or 13C nuclei are predominantly used for the characterization of the protein of interest. These measurements are limited due to the wealth of chemical shifts and background signals arising from all molecules present in the NMR test tube. On top of that, the protein under study has to be isotopically enriched in nitrogen and/or carbon nuclei enabling to overcome the inherently low natural abundance of 13C and 15N NMR active isotopes. In this way switching to 19F NMR spectroscopy strongly reduces the total amount of signals seen in an NMR spectrum as it turns off background signals and is for this reason extremely attractive for highly-resolved investigations of proteins performance measured directly in cells or in a cell-like environment. Here we show the effective expression and purification of cold shock protein B from Bacillus subtilis (BsCspB) using fluorine labelled phenylalanine or fluorine labelled tryptophan residues. We reveal that fluorine labelled BsCspB represents the same fold on a secondary as tertiary level as seen for the wild type protein independent of the labelling position illuminating the soft character of fluorine insertion. This experimental setup of targeted fluorine labelling sets a profound ground for a broad range of highly-resolved 19F NMR applications to be performed in a complex cellular environment.publishe