8,964 research outputs found
Heuristic Refinement Method for the Derivation of Protein Solution Structures: Validation on Cytochrome B562
A method is described for determining the family of protein structures compatible with solution data obtained primarily from nuclear magnetic resonance (NMR) spectroscopy. Starting with all possible conformations, the method systematically excludes conformations until the remaining structures are only those compatible with the data. The apparent computational intractability of this approach is reduced by assembling the protein in pieces, by considering the protein at several levels of abstraction, by utilizing constraint satisfaction methods to consider only a few atoms at a time, and by utilizing artificial intelligence methods of heuristic control to decide which actions will exclude the most conformations. Example results are presented for simulated NMR data from the known crystal structure of cytochrome b562 (103 residues). For 10 sample backbones an average root-mean-square deviation from the crystal of 4.1 A was found for all alpha-carbon atoms and 2.8 A for helix alpha-carbons alone. The 10 backbones define the family of all structures compatible with the data and provide nearly correct starting structures for adjustment by any of the current structure determination methods
The challenges of purely mechanistic models in biology and the minimum need for a 'mechanism-plus-X' framework
Ever since the advent of molecular biology in the 1970s, mechanical models have become the dogma in the field, where a "true" understanding of any subject is equated to a mechanistic description. This has been to the detriment of the biomedical sciences, where, barring some exceptions, notable new feats of understanding have arguably not been achieved in normal and disease biology, including neurodegenerative disease and cancer pathobiology. I argue for a "mechanism-plus-X" paradigm, where mainstay elements of mechanistic models such as hierarchy and correlation are combined with nomological principles such as general operative rules and generative principles. Depending on the question at hand and the nature of the inquiry, X could range from proven physical laws to speculative biological generalizations, such as the notional principle of cellular synchrony. I argue that the "mechanism-plus-X" approach should ultimately aim to move biological inquiries out of the deadlock of oft-encountered mechanistic pitfalls and reposition biology to its former capacity of illuminating fundamental truths about the world
Frustration in Biomolecules
Biomolecules are the prime information processing elements of living matter.
Most of these inanimate systems are polymers that compute their structures and
dynamics using as input seemingly random character strings of their sequence,
following which they coalesce and perform integrated cellular functions. In
large computational systems with a finite interaction-codes, the appearance of
conflicting goals is inevitable. Simple conflicting forces can lead to quite
complex structures and behaviors, leading to the concept of "frustration" in
condensed matter. We present here some basic ideas about frustration in
biomolecules and how the frustration concept leads to a better appreciation of
many aspects of the architecture of biomolecules, and how structure connects to
function. These ideas are simultaneously both seductively simple and perilously
subtle to grasp completely. The energy landscape theory of protein folding
provides a framework for quantifying frustration in large systems and has been
implemented at many levels of description. We first review the notion of
frustration from the areas of abstract logic and its uses in simple condensed
matter systems. We discuss then how the frustration concept applies
specifically to heteropolymers, testing folding landscape theory in computer
simulations of protein models and in experimentally accessible systems.
Studying the aspects of frustration averaged over many proteins provides ways
to infer energy functions useful for reliable structure prediction. We discuss
how frustration affects folding, how a large part of the biological functions
of proteins are related to subtle local frustration effects and how frustration
influences the appearance of metastable states, the nature of binding
processes, catalysis and allosteric transitions. We hope to illustrate how
Frustration is a fundamental concept in relating function to structural
biology.Comment: 97 pages, 30 figure
Changes in cardiomyocyte structure and cAMP/cGMP signalling during heart failure
The contractile function of the heart depends on efficient β adrenergic receptor (βAR) signalling which involves cycling nucleotides as second messengers. Correct secondary messenger signalling is only possible in healthy, well structured cardiac myocytes. Of the three βAR subtypes present in human cardiomyocytes β1AR and β2AR classically signal via 3'-5' cyclic adenosine monophosphate (cAMP) to regulate contraction after catecholamine administration, whereby the second isoform may also be cardioprotective. The far less characterised β3AR has been controversially associated to both increasing contraction through cAMP and protecting the heart through 3'-5' cyclic guanosine monophosphate (cGMP) signalling. During the progression of heart failure following myocardial infarction (MI) both the normal cell structure and the regulation of cAMP and cGMP signalling are changed. This happens in part due to changes in catecholaminergic stimulation of the βARs and in mechanical load, as well as due to a progressive development of hypertrophy. Some of the alterations initially appear to be of a compensatory nature but escalate into HF by worsening cardiomyocyte function and cell survival.
The work presented here (1) investigates the structural integrity of healthy, isolated, single cardiomyocytes by looking at the surface topography via Scanning Ion Conductance Microscopy (SICM) imaging and by examining the internal Transverse Axial Tubule (TAT) network via confocal imaging; (2) elucidates the cyclic nucleotide response to catecholamine stimulation following either global (in the solution) or local (in the SICM pipette) stimulation of either β2ARs or β3ARs and measuring either cAMP or cGMP levels via FÜrster Resonance Energy Transfer (FRET) sensors in a combined FRET/SICM imaging setup; (3) determines how both the structure and β2AR and β3AR dependent second messenger signalling change in a progressive rat model of HF 4, 8 and 16 weeks after the induction of chronic MI.
The major findings of the presented work are as follows:
In control cardiomyocytes the structure is highly intricate with regular Z-grooves and crest areas. In MI cells the normal suface topography progressively deteriorates, with the eventual disappearance of Z-grooves by week 16, which correlates with the disorganisation of the cardiomyocyteâs internal transverse axial tubule (TAT) network of T-tubules emanating from the cell surface and traversing into the cell centre. This is accompanied by the gradual redistribution of β2ARs from their normal position inside the T-tubules to the unstructured areas on the cardiomyocyte membrane. The regularity and density of the TAT network is already severely compromised at 4 weeks post MI; at the same time a significant drop in the expression of the structural protein Junctophilin 2 (JPH2) occurs. At 4 and 8 weeks post MI a potentially compensatory increase in the number of longitudinal elements takes place which was no longer detectable at 16 weeks. The production of cAMP following local stimulation of β2ARs in the T-tubule openings was already suppressed at 4 weeks post MI and a β2AR response becomes detectable after local stimulation at the cell crests (areas between Z-grooves) at 8 weeks post MI. At 16 weeks post MI the β2AR-dependent cAMP level following both global and local stimulations was reduced due to an overall decrease in the adenylate cyclase (AC) activity.
The production of the second cyclic nucleotide, cGMP, following β3AR stimulation is evident in control cells and to a significantly lesser extent in myocytes isolated from hearts at the end stage of HF. These β3AR-cGMP levels were degraded mainly by phosphodiesterases (PDE) 2 and 5. Local stimulation through the SICM pipette reveals that functional β3ARs are primarily localized inside T-tubules in control cells but redistribute equally in between T-tubules and crests in cells isolated from failing hearts.
To improve the accuracy and reliability of local application of agonists via the SICM nanopipette voltage was applied to the pipette, as opposed to previously employed displacement of the liquid in the pipette via air pressure. Mathematical modelling served to determine the correct settings for this voltage driven application. It shows that the SICM nanopipette can reliably and precisely unload the βAR agonist ISO onto the nanoscale structure of cardiomyocytes via voltage.Open Acces
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