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The Physiological Molecular Shape of Spectrin: A Compact Supercoil Resembling a Chinese Finger Trap.

Brown JW, Bullitt E, Sriswasdi S, Harper S, Speicher DW, McKnight CJ - PLoS Comput. Biol. (2015)

Bottom Line: We validated our model with electron microscopy, which demonstrated that, as predicted, spectrin is hollow at its biological resting length of ~55-65 nm.The model is further supported by zero-length chemical crosslink data indicative of an approximately 90 degree bend between adjacent spectrin repeats.The domain-domain interactions in our model are entirely consistent with those present in the prototypical linear antiparallel heterotetramer as well as recently reported inter-strand chemical crosslinks.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts, United States of America; Internal Medicine Residency Program, University of Pittsburgh Medical Center, UPMC Montefiore Hospital, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
The primary, secondary, and tertiary structures of spectrin are reasonably well defined, but the structural basis for the known dramatic molecular shape change, whereby the molecular length can increase three-fold, is not understood. In this study, we combine previously reported biochemical and high-resolution crystallographic data with structural mass spectroscopy and electron microscopic data to derive a detailed, experimentally-supported quaternary structure of the spectrin heterotetramer. In addition to explaining spectrin's physiological resting length of ~55-65 nm, our model provides a mechanism by which spectrin is able to undergo a seamless three-fold extension while remaining a linear filament, an experimentally observed property. According to the proposed model, spectrin's quaternary structure and mechanism of extension is similar to a Chinese Finger Trap: at shorter molecular lengths spectrin is a hollow cylinder that extends by increasing the pitch of each spectrin repeat, which decreases the internal diameter. We validated our model with electron microscopy, which demonstrated that, as predicted, spectrin is hollow at its biological resting length of ~55-65 nm. The model is further supported by zero-length chemical crosslink data indicative of an approximately 90 degree bend between adjacent spectrin repeats. The domain-domain interactions in our model are entirely consistent with those present in the prototypical linear antiparallel heterotetramer as well as recently reported inter-strand chemical crosslinks. The model is consistent with all known physical properties of spectrin, and upon full extension our Chinese Finger Trap Model reduces to the ~180-200 nm molecular model currently in common use.

No MeSH data available.


Related in: MedlinePlus

Electron microscopy of actin-spectrin ladders.A. Transmission electron micrograph (TEM) of negatively-stained actin and spectrin illustrating “ladders”, with actin as the ‘rails’ and spectrin as the ‘rungs’. B. Cartoon of actin and spectrin overlaid on the micrograph shown in panel A (actin: white, spectrin: black; not to scale). C and D. TEM and cartoon, respectively, of frozen-hydrated actin and spectrin, again illustrating the ladder-like appearance. Contrast of the cryoEM images is inverted so that protein is white, as in panels A, B and E. E. TEMs of negatively stained spectrin viewed end-on, in which some images show a hollow center. F. Radial density profile of a representative end-on view of spectrin; note that the center density (at r = 0) is low, and rises before falling off to the background value.
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pcbi.1004302.g005: Electron microscopy of actin-spectrin ladders.A. Transmission electron micrograph (TEM) of negatively-stained actin and spectrin illustrating “ladders”, with actin as the ‘rails’ and spectrin as the ‘rungs’. B. Cartoon of actin and spectrin overlaid on the micrograph shown in panel A (actin: white, spectrin: black; not to scale). C and D. TEM and cartoon, respectively, of frozen-hydrated actin and spectrin, again illustrating the ladder-like appearance. Contrast of the cryoEM images is inverted so that protein is white, as in panels A, B and E. E. TEMs of negatively stained spectrin viewed end-on, in which some images show a hollow center. F. Radial density profile of a representative end-on view of spectrin; note that the center density (at r = 0) is low, and rises before falling off to the background value.

Mentions: The molecular shape of spectrin was further analyzed using transmission electron microscopy (EM). Because we found spectrin to stain very poorly with several common contrast agents, we co-incubated spectrin with actin to better delineate the protein. The combination of these two proteins resulted in the formation of a ladder-like configuration that was visible in both negatively stained samples using EM (Fig 5A–5B), and unstained samples viewed using cryoEM (Fig 5C–5D). In these images, actin filaments are the rails and spectrin tetramers form the rungs of a ladder; this interpretation is supported by the presence of one spectrin binding site for each actin monomer, allowing numerous spectrin molecules to bind per filament, whereas spectrin has two F-actin binding sites so that one heterotetramer of spectrin can bind two actin filaments. Similar to EM examination of native preparations of spectrin [14–17], we found spectrin to be linear over a wide range of lengths (Fig 5).


The Physiological Molecular Shape of Spectrin: A Compact Supercoil Resembling a Chinese Finger Trap.

Brown JW, Bullitt E, Sriswasdi S, Harper S, Speicher DW, McKnight CJ - PLoS Comput. Biol. (2015)

Electron microscopy of actin-spectrin ladders.A. Transmission electron micrograph (TEM) of negatively-stained actin and spectrin illustrating “ladders”, with actin as the ‘rails’ and spectrin as the ‘rungs’. B. Cartoon of actin and spectrin overlaid on the micrograph shown in panel A (actin: white, spectrin: black; not to scale). C and D. TEM and cartoon, respectively, of frozen-hydrated actin and spectrin, again illustrating the ladder-like appearance. Contrast of the cryoEM images is inverted so that protein is white, as in panels A, B and E. E. TEMs of negatively stained spectrin viewed end-on, in which some images show a hollow center. F. Radial density profile of a representative end-on view of spectrin; note that the center density (at r = 0) is low, and rises before falling off to the background value.
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pcbi.1004302.g005: Electron microscopy of actin-spectrin ladders.A. Transmission electron micrograph (TEM) of negatively-stained actin and spectrin illustrating “ladders”, with actin as the ‘rails’ and spectrin as the ‘rungs’. B. Cartoon of actin and spectrin overlaid on the micrograph shown in panel A (actin: white, spectrin: black; not to scale). C and D. TEM and cartoon, respectively, of frozen-hydrated actin and spectrin, again illustrating the ladder-like appearance. Contrast of the cryoEM images is inverted so that protein is white, as in panels A, B and E. E. TEMs of negatively stained spectrin viewed end-on, in which some images show a hollow center. F. Radial density profile of a representative end-on view of spectrin; note that the center density (at r = 0) is low, and rises before falling off to the background value.
Mentions: The molecular shape of spectrin was further analyzed using transmission electron microscopy (EM). Because we found spectrin to stain very poorly with several common contrast agents, we co-incubated spectrin with actin to better delineate the protein. The combination of these two proteins resulted in the formation of a ladder-like configuration that was visible in both negatively stained samples using EM (Fig 5A–5B), and unstained samples viewed using cryoEM (Fig 5C–5D). In these images, actin filaments are the rails and spectrin tetramers form the rungs of a ladder; this interpretation is supported by the presence of one spectrin binding site for each actin monomer, allowing numerous spectrin molecules to bind per filament, whereas spectrin has two F-actin binding sites so that one heterotetramer of spectrin can bind two actin filaments. Similar to EM examination of native preparations of spectrin [14–17], we found spectrin to be linear over a wide range of lengths (Fig 5).

Bottom Line: We validated our model with electron microscopy, which demonstrated that, as predicted, spectrin is hollow at its biological resting length of ~55-65 nm.The model is further supported by zero-length chemical crosslink data indicative of an approximately 90 degree bend between adjacent spectrin repeats.The domain-domain interactions in our model are entirely consistent with those present in the prototypical linear antiparallel heterotetramer as well as recently reported inter-strand chemical crosslinks.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts, United States of America; Internal Medicine Residency Program, University of Pittsburgh Medical Center, UPMC Montefiore Hospital, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
The primary, secondary, and tertiary structures of spectrin are reasonably well defined, but the structural basis for the known dramatic molecular shape change, whereby the molecular length can increase three-fold, is not understood. In this study, we combine previously reported biochemical and high-resolution crystallographic data with structural mass spectroscopy and electron microscopic data to derive a detailed, experimentally-supported quaternary structure of the spectrin heterotetramer. In addition to explaining spectrin's physiological resting length of ~55-65 nm, our model provides a mechanism by which spectrin is able to undergo a seamless three-fold extension while remaining a linear filament, an experimentally observed property. According to the proposed model, spectrin's quaternary structure and mechanism of extension is similar to a Chinese Finger Trap: at shorter molecular lengths spectrin is a hollow cylinder that extends by increasing the pitch of each spectrin repeat, which decreases the internal diameter. We validated our model with electron microscopy, which demonstrated that, as predicted, spectrin is hollow at its biological resting length of ~55-65 nm. The model is further supported by zero-length chemical crosslink data indicative of an approximately 90 degree bend between adjacent spectrin repeats. The domain-domain interactions in our model are entirely consistent with those present in the prototypical linear antiparallel heterotetramer as well as recently reported inter-strand chemical crosslinks. The model is consistent with all known physical properties of spectrin, and upon full extension our Chinese Finger Trap Model reduces to the ~180-200 nm molecular model currently in common use.

No MeSH data available.


Related in: MedlinePlus