Biology subjects

Jacobsen, N. L.

Publications and source records attributed to Jacobsen, N. L..

4 recordsLinked to original sources

bioRxiv2023

Revisiting the use of dioxane as a reference compound for determination of the hydrodynamic radius of proteins by pulsed field gradient NMR spectroscopy

Measuring the compaction of a protein or complex is key to understand the interactions within and between biomolecules. Experimentally, protein compaction is often probed either by estimating the radius of gyration (Rg) obtained from small-angle X-ray scattering (SAXS) experiments or the hydrodynamic radius (Rh) obtained for example by pulsed field gradient nuclear magnetic resonance (PFG NMR) spectroscopy. PFG NMR experiments generally report on the translational diffusion coefficient, which in turn can be used to estimate Rh using an internal standard. Here, we examine the use of 1,4-dioxane as an internal NMR standard to account for sample viscosity and uncertainty about the gradient strength. Specifically, we revisit the basis for the commonly used reference value for the Rh of dioxane (2.12 [A]) that is used to convert measured diffusion coefficients into a hydrodynamic radius. We follow the same approach that was used to establish the current reference value for the Rh by measuring SAXS and PFG NMR data for a set of seven different proteins and using these as standards. Our analysis shows that the current Rh reference value for 1,4-dioxane Rh (2.12 [A]) is underestimated, and we instead suggest a new value of 2.27 [A] {+/-} 0.04 [A]. Using this updated reference value results in a [~]7% increase in Rh values for proteins whose hydrodynamic radius have been measured by PFG NMR. We discuss the implications for ensemble descriptions of intrinsically disordered proteins and evaluation of effect resulting from for example ligand binding, posttranslational modifications, or changes to the environment.

biophysics↗

bioRxiv2022

Deciphering the alphabet of disorder -- Glu and Asp act differently on local but not global properties

Compared to folded proteins, the sequences of intrinsically disordered proteins (IDPs) are enriched in polar and charged amino acids. Glutamate is one of the most enriched amino acids in IDPs, while the chemically similar amino acid aspartate is less enriched. So far, the underlying functional differences of glutamates and aspartates in IDPs remain poorly understood. In this study, we examine the differential effects of aspartate and glutamates in IDPs by comparing the function and conformational ensemble of glutamate and aspartate variants of the disordered protein Dss1, using a range of assays, including interaction studies, nuclear magnetic resonance spectroscopy, small angle X-ray scattering and molecular dynamics simulation. First, we analyze the sequences of the rapidly growing data base of experimentally verified IDPs (DisProt) and show that the glutamate enrichment is not caused by a taxonomy bias in IDPs. From analyses of local and global structural properties as well as cell growth and protein-protein interactions using a model acidic IDP from yeast and three Glu/Asp variants, we find that while Glu/Asp support similar function and global dimensions, the variants differ in their binding affinities and population of local transient structural elements. We speculate that these local structural differences may play roles in functional diversity where glutamates can support increased helicity important for folding and binding, while aspartates support extended structures and form helical caps, as well as playing more relevant roles in e.g., transactivation domains and ion-binding.

biophysics↗

bioRxiv2022

Angiogenesis precedes myogenesis during regenerationfollowing biopsy injury in skeletal muscle

BackgroundAcute injury to skeletal muscle damages myofibers and fragments capillaries, impairing contractile function and local perfusion. Myofibers and microvessels regenerate from satellite cells and from surviving microvessel fragments, respectively, to restore intact muscle. However, it is unknown whether myofiber regeneration and microvascular regeneration reflect interdependent processes or may proceed sequentially. MethodsTo investigate the temporal relationship between myogenesis and angiogenesis during regeneration, a punch biopsy (diameter, 2 mm) was performed through the center of the gluteus maximus (GM) muscle. Complete removal of all tissue components created a void into which regeneration was evaluated through 21 days post injury (dpi). Confocal imaging and histological analyses of whole-mount GM preparations and GM cross sections assessed the growth of microvessels and myofibers into the wound. Regeneration of perfused microvessels was evaluated in vivo by injecting fluorescent dextran into the circulation during intravital imaging. ResultsA provisional matrix filled with PDGFR+ and CD45+ cells spanned the wound within 1 dpi. Regenerating microvessels advanced into the matrix by 7 dpi. At 10 dpi, sprouting and intussusceptive angiogenesis produced disorganized microvascular networks and spanned the wound with perfusion by 14 dpi. In striking contrast, the wound remained devoid of myofibers at 7 and 10 dpi. Myogenesis into the wound began by 14 dpi with nascent myofibers traversing the wound by 21 dpi. Regenerating myofibers and microvessels were less well organized than in the surrounding (uninjured) muscle. ConclusionsAngiogenesis precedes myogenesis following punch biopsy injury of adult skeletal muscle. Regenerating microvessels encompass the wound and become perfused with blood prior to colocalization with regenerating myofibers. These findings infer that a microvascular supply supports the metabolic demands of regenerating skeletal muscle. Finding that regenerated microvascular networks and myofibers are disorganized within the biopsy site suggests that loss of guidance cues upon complete tissue removal impairs re-establishment of canonical skeletal muscle structure.

physiology↗

bioRxiv2021

Myofiber injury induces capillary disruption and regeneration of disorganized microvascular networks

Myofibers regenerate following injury, however the microvasculature must also recover to restore skeletal muscle function. We aimed to define the nature of microvascular damage and repair during skeletal muscle injury and regeneration induced by BaCl2. To test the hypothesis that microvascular disruption occurred secondary to myofiber injury in mice, isolated microvessels were exposed to BaCl2 or the myotoxin was injected into the gluteus maximus (GM) muscle. In isolated microvessels, BaCl2 depolarized smooth muscle cells and endothelial cells while increasing [Ca2+]i, but did not elicit cell death. At 1 day post injury (dpi) of the GM, capillary fragmentation coincided with myofiber degeneration while arteriolar and venular networks remained intact; neutrophil depletion before injury did not prevent capillary damage. Perfused capillary networks reformed by 5 dpi in association with more terminal arterioles and were dilated through 10 dpi; with no change in microvascular area or branch point number in regenerating networks, fewer capillaries aligned with myofibers and capillary networks were no longer organized into microvascular units. By 21 dpi, capillary orientation and organization had nearly recovered to that in uninjured GM. We conclude that following their disruption secondary to myofiber damage, capillaries regenerate as disorganized networks that remodel while regenerated myofibers mature.

physiology↗