Dilated cardiomyopathy (DCM) is a serious, and poorly understood, heart disease that affects about 1 million people in the United State. DCM is a progressive disease with no current cure, often culminating in heart transplantation. Many cases of DCM are caused by inherited gene mutations often located on specific muscle proteins that are part of the cell machinery that allows contractions. One such protein is Myosin Light Chain 2 (MYL2), part of the “motor” that powers contraction. The investigators identified a human mutation that causes DCM called D94A, and created a transgenic mouse that expresses this mutation allowing the mouse heart muscle to be studied by a wide range of techniques including X-ray diffraction at the BioCAT Beamline 18ID at the Advanced Photon Source, Argonne National Laboratory. The X-ray experiments showed that one of the reasons the muscle does not contract correctly is that the myosin ”motor” proteins are positioned further away from their targets than in normal heart muscle making it harder for them to generate the correct amounts of force to pump the right amount of blood …

DNA in eukaryotic cells is normally found associated with protiens called histone in particles called nucleosomes linked by short segments of DNA. Chromatin remodelers are specialized ATP-dependent molecular machines that can reorganize the structure of nucelsomes as needed for such processes such as replication, transcription and DNA repair.

Remodeling results in evenly spaced nucleosomes along the DNA strand yet the way remodelers achieve this is not understood. CHD-1 is a remodeler important for transcription. Here, the authors show that the Chd1 remodeler shifts DNA back and forth by dynamically alternating between different segments of the nucleosome. During sliding, Chd1 generates unstable remodeling intermediates that spontaneously relax to a pre-remodeled position. They demonstrate that nucleosome sliding is tightly controlled by two regulatory domains: the DNA binding domain, which interferes with sliding when its range is limited by a truncated linking segment, and the chromodomains, which play a key role in substrate discrimination. They propose that active interplay of the ATPase motor with the regulatory domains may promote dynamic nucleosome structures uniquely suited for histone exchange and chromatin reorganization during transcription. This work advances our understanding of the Chd1 chromatin remodeler, and puts forward several concepts that may also apply …

The physical basis of protein folding stability and cooperativity remains a topic of great interest. Folding of globular proteins is generally assumed to be driven by energetically favorable burial of hydrophobic groups and that early development of secondary structure increases the cooperativity of folding. The Sosnick group at the University of Chicago examines these assumptions in a protein (snow flea antifreeze protein (sfAFP) that is striking in its dearth of hydrophobic burial and its lack of α and β structures, while having a low sequence complexity with 46% glycine. The interior of the protein is filled only with backbone H-bonds between six polyproline 2 (PP2) helices. Unexpectedly, the protein folds in a kinetically two-state manner and is moderately stable at room temperature, similar behavior to that observed for typical globular proteins having α and β structures and a hydrophobic core. Hence, these features are not necessary for folding cooperativity and stability. This enigma forces a reexamination of the possible combination of factors that can stabilize a protein. The authors propose that a major part of the stability arises from the unusual match between residue-level PP2 dihedral angle bias in the unfolded state and PP2 helical structure in the native state …

In eukaryotic cells, diverse stresses trigger coalescence of various RNA binding proteins into so-called stress granules. In vitro, stress-granule associated proteins have been observed to demix to form liquids, hydrogels, and other assemblies. Demixing of an abundant RNA-binding protein into hydrogel droplets, triggered by stress-associated physiological conditions, appears to promote cell fitness during stress.

Here, a U Chicago based team lead by D. Allan Drummond and Tobin Sosnick showed that poly(A)-binding protein (Pab1 in yeast), a defining marker of stress granules, phase separates and forms hydrogels in vitro upon exposure to physiological stress conditions. Other RNA-binding proteins depend upon low-complexity regions (LCRs) or RNA for phase separation. Based on unique evolutionary patterns, the authors created LCR mutations, which systematically tune its biophysical properties and Pab1 phase separation in vitro and in vivo. Mutations that impede phase separation reduced organism fitness during prolonged stress. Such mutations reduce the thermal and pH sensitivity of Pab1’s demixing and, hence, reduce fitness during growth at high temperature and during energy depletion, indicating that demixing is adaptive. Together, the results illuminate a uniquely complete path from evolved sequence features, to phase separation, to stress-triggered demixing, and finally to organism fitness …

Chloroplasts, like mitochondria, are organelles of endosymbiotic origin, having evolved from initial engulfment of a cyanobacterium by a eukaryotic cell. Most of the bacterial genome was subsequently lost so that most proteins found within chloroplasts are synthesized in the cytoplasm as preproteins and then imported via specialized machinery prior to trafficking to their final destination. Protein import is accomplished by the TOC (translocon on the outer chloroplast membrane) and TIC (translocon on the inner chloroplast membrane) machineries in the outer and inner envelope membranes, respectively. The TOC complex includes a protein called Toc75, which serves as the translocation channel along with two other proteins, as well as Toc33 and Toc159, which both contain GTPase domains, which help drivesubstrate selection and importation. Structural information for the TOC complex was hitherto lacking, hindering the ability of investigators to form mechanistic models for function. Here a team lead by Nicholas Noinaj (Purdue University) and Danny Schnell (Michigan State University) reported crystals structures of Toc75 consisting of three tandem POTRA domains. High quality size exclusion chromatography coupled SAXS experiments at the BioCAT Beamline 18ID were important in establishing that crystals structures accurately represented protein structure in solution. The POTRA domains may help facilitate preprotein …

Human immunodeficiency virus type 1 (HIV-1) requires controlled synthesis of its protein complement for persistent infection and successful virion production. Genome expression is tightly regulated at the levels of transcription, splicing, mRNA nuclear export, and translation. RNA polymerase II-dependent transcription yields a 9-kilobase (kb) polycistronic transcript that undergoes multiple rounds of alternative splicing to produce upward of 100 different viral mRNAs that recruit antagonistic host RNA-binding proteins. Thus, HIV-1 splicing pathways are essential components of the viral replication cycle and represent new targets for therapeutic intervention. Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. SEC-SAXS data collected at BioCAT was used to confirm the general features of the NMR structure and by refining the NMR structure against the SAXS data, obtain a better structure than that of NRM alone. Collectively, this work provides additional insights into how HIV-1 uses a …