Understanding the driving forces behind stability of denatured state ensembles (DSE’s) and intrinsically disordered proteins (IDP’s) is central to a number of unresolved questions in bimolecular thermodynamics regarding protein folding pathways and foldability, thermodynamic stability, aggregation and misfolding. Researchers at the University of Chicago and Notre Dame used temperature-controlled size-exclusion chromatography-coupled SAXS (SEC-SAXS) and NMR to examine how temperature and solvent ionic strength influences the solution structure(s) of the N-terminal domain of pertactin (PNt). PNt is a valuable model system from a fundamental biophysical point of view, as the full-length 539-residue pertactin folds into a parallel β-helix but the 334 N-terminal residues do not and instead behave as a highly expanded, intrinsically disordered chain.

In this work, the authors used temperature-series SEC-SAXS experiments performed at BioCAT, with temperatures ranging from 4 to 60 degrees C, to show that PNt displays a mild, temperature-dependent contraction (as measured by R_g value). Glycine-rich low hydrophobicity model systems (as proxies for protein backbones alone) did not display an analogous trend, showing the specific importance of side chain interactions. Increasing the ionic strength of the …

The Frank-Starling Law of the Heart states that the heart’s stroke volume increases with greater preload due to increased venous return, allowing the heart to adapt to varying circulatory demands. At the molecular level, increasing preload increases sarcomere length (SL), which alters structures within the sarcomere that are correlated to increased calcium sensitivity upon activation. The titin protein, spanning the half-sarcomere acts as a spring in the I-band, applies a SL-dependent passive force on the myosin containing thick filaments changing its structure and functional properties. Altered titin-based forces play a crucial role in the etiology of many cardiomyopathies; however, the disease state obscures titin’s role, impeding therapeutic solutions.

The authors studied titin’s specific role using the titin cleavage (TC) mouse model, where a tobacco-etch virus protease (TEVP) recognition site is inserted into distal I-band titin and allows for rapid, specific cleavage …

Muscle contraction is produced via the interaction of myofilaments and is regulated so that muscle performance matches demand. Myosin-binding protein C (MyBP-C) is a long and flexible protein that is tightly bound to the thick filament at its C-terminal end (MyBP-C^C8C10), but may be loosely bound at its middle- and N-terminal end (MyBP-C^C1C7) to myosin heads and/or the thin filament. MyBP-C is thought to control muscle contraction via the regulation of myosin motors, as mutations lead to debilitating disease. Thick filament based regulatory mechanisms involve either sequestering myosin head into an inactive ordered off state close to the thick filament backbone or releasing them so that they can interact with actin. Strain in the thick filaments, either from a few active heads or passively generated by stretching titin, is presumed to act as a trigger to release inactive heads. While MyBP-C has been presumed to play a role(s) in thick filament activation, details of these mechanisms have been lacking. Here the authors used combination of mechanics and small-angle X-ray diffraction to study the effects of immediate and selective removal of the MyBP-C^C1C7 domains of fast MyBP-C on sarcomere structure and function in permeabilized skeletal muscle …

Netrins are a conserved class of proteins involved in synaptic connectivity of the nervous system in bilaterian animals. They act as secreted guidance cues, with the unique ability to exert repulsive and attractive responses on growing axons. They are also known to be involved in cell proliferation, migration and differentiation, and are therefore targets for treating cancer and insulin resistance. During axon growth and cell migration, the presence of the receptor Uncoordinated-5 (UNC-5) on target cells results in repulsion. However, the exact mechanism involved in the induction of repulsive forces has been relatively unknown due to the lack of biochemical and structural information about these systems. Researchers at the University of Chicago and Stanford University, in collaboration with BioCAT staff, showed that UNC-5 is a heparin-binding protein, determined its structure bound to a heparin fragment, and could modulate UNC-5–heparin affinity using a directed evolution platform or structure-based rational design.

A question they needed to answer was whether the heparin + UNC-6–mediated UNC-5 oligomers are an ordered and well-defined molecular species or (ii) whether these oligomers …

Mavacamten is the first myosin-targeted small-molecule therapy approved by the Food and Drug Administration to treat obstructive hypertrophic cardiomyopathy by attenuating excessive myocardial sarcomere activity. Mavacamten regulates cardiac function at the sarcomere level by selectively but reversibly inhibiting the enzymatic activity of myosin. It shifts myosin toward ordered off states close to the thick filament backbone making them unavailable for binding to actin and generating force. It is necessary, however for the heart to adjust its output to ensure sufficient cardiac output, especially during increased physiological demands. It was unknown whether mavacamten stabilized heads could still be recruited by the usual physiological inotropic mechanisms for the patient to be able to adapt to changing demands on their hearts …

Myosin-based thick-filament regulation is now known to be critical for muscle contraction with myosin dysregulation found in hypertrophic and dilated cardiomyopathies but many details of thick filament regulation remain to be discovered. Myosin ATPase assays have demonstrated that under relaxed conditions, myosin may reside either in a high-energy-consuming disordered-relaxed (DRX) state available for binding actin to generate force or in an energy-sparing super-relaxed (SRX) state unavailable for actin binding. X-ray diffraction studies have shown that the majority of myosin heads are in a …

Autophagy is a conserved lysosomal degradation pathway that degrades un-needed cellular components such as misfolded, aggregated, mutated and damaged proteins, organelles, and pathogens. Autophagy dysfunction is implicated in numerous diseases including neurodegenerative disorders, muscular diseases, cardiomyopathy, cancer and infectious diseases. Many proteins involved in autophagy contain intrinsically disordered regions (IDRs) that do not form stable secondary or tertiary structure. The structural flexibility of IDRs is thought to enable diverse and multiple interactions enabling them to regulate cell signaling pathways. Many IDRs have been shown to fold upon binding to ligands. BECN1, a key autophagy protein involved in autophagosome nucleation, contains two invariant CxxC motifs within a large BECN1 intrinsically disordered region (IDR) at the BECN1 N-terminus. The goal of the research was to uncover the functional roles of the invariant CxxC motifs which were hitherto not understood. The authors used inductively coupled plasma mass spectrometry to demonstrate that BECN1 binds Zn²⁺ in a 1:1 molar ratio, and that mutation of the invariant cysteines prevents co-ordination of Zn²⁺, demonstrating that the CxxC motifs are responsible for binding Zn²⁺. Zn^{2 …}

Semaglutide (SMG) is class of modified, acetylated peptide mimic commonly used as a commercial therapeutic to treat type-2 diabetes and obesity. Like other classes of peptide mimic therapeutics, SMG’s suffer from physical instabilities, including various aggregation and degradation pathways but also spontaneous emulsification into colloidal structures in the presence of certain hydrophobic surfaces, a process often termed “ouzo formation.” Researchers at the University of Delaware Center for Neutron Science, in collaboration with Eli Lilly, used a variety of biophysical methods including small-angle X-ray scattering (SAXS), circular dichroism (CD) and dynamic light scattering (DLS) to elucidate the fundamental physical mechanisms behind ouzo formation. SAXS experiments at BioCAT indicated that the colloidal droplets consisted of aggregated oligomers, which themselves form as a result of self-association of the peptides. Together with other experiments, the authors propose a mechanism where initial nucleation is catalyzed by adsorption of oligomers with a hydrophobic surface, where the effects of specific surfaces can be correlated with Hansen Solubility Parameters. Following nucleation (and a subsequent desorption step), nucleated droplets grow rapidly, ultimately resulting …

The ways in which starches, in particular digestion-resistant starches, are accommodated by gut bacteria remains relatively poorly understood at the molecular level. Digestion-resistant starches are accessed by specialized gut bacteria with specific carbohydrate-binding systems. One such system is the Ruminococcus bromii Starch Adherence System member 6 (Sas6), which consists of two starch-specific carbohydrate-binding modules from family 26 (RbCBM26) and family 74 (RbCBM74). The authors present a structural and functional characterization by crystallography, SAXS, native mass spec and other methods of one such system from Ruminococcus bromii (Sas6). The crystal structure of Sas6 showed that the RbCBM74 starch-binding groove complements the double helical α-glucan geometry of amylopectin, suggesting that this module selects this feature in starch granules. The overall structure was compact – both in crystals and in solution, as measured by size-exclusion chromatography-coupled SAXS (SEC-SAXS) – stabilized by significant hydrogen bonding networks between the domains. SEC-SAXS experiments …

Biomolecular condensates are involved in a range of cellular processes including stress response, protein degradation and gene expression. These condensates contain a wide range of unique macromolecules, but the drivers of this condensation, referred to as scaffolds, comprise only a very small fraction. The non-driver components are commonly referred to as ligands and may not phase separate on their own but nonetheless may help regulate assembly, disassembly and other material properties. One such ligand is ubiquitin (Ub) or its linked multimers (polyubiquitin chains), which are attached as posttranslational modifications to partner proteins and help determine various downstream signaling outcomes such as DNA repair. There is growing evidence that suggests the involvement of polyUb chains in phase separation acts as a mechanism for the reading and interpretation of said Ub code in the cell. Research from the Castaneda lab at Syracuse University has worked towards understanding the molecular rules by which polyUb chains …