SAXS analysis of a soluble cytosolic NgBR construct including extracellular and transmembrane domains

Autor: Emerson Perry, Yingxue Zhang, Lindsey Nico, Nicholas Spellmon, Weifeng Shang, Qing Miao, Srinivas Chakravarthy, Junmei Wan, Timothy L. Stemmler, Brianne E. Lewis, Maysaa Doughan, Joshua Holcomb, Zhe Yang
Jazyk: angličtina
Rok vydání: 2018
Předmět:
0301 basic medicine
Circular dichroism
Cell Membranes
lcsh:Medicine
Endoplasmic Reticulum
Biochemistry
Protein Structure
Secondary
Small-Angle Scattering
Scattering
Protein structure
Cytosol
Spectrum Analysis Techniques
X-Ray Diffraction
lcsh:Science
Peptide sequence
Protein secondary structure
Multidisciplinary
Crystallography
Secretory Pathway
Chemistry
Small-angle X-ray scattering
Circular Dichroism
Physics
Chromatographic Techniques
Condensed Matter Physics
Recombinant Proteins
Transmembrane domain
Circular Dichroism Spectroscopy
Cell Processes
Physical Sciences
Radius of gyration
Crystal Structure
Cellular Structures and Organelles
Research Article
Size-Exclusion Chromatography
Receptors
Cell Surface
Research and Analysis Methods
03 medical and health sciences
Scattering
Small Angle
Solid State Physics
Amino Acid Sequence
Binding site
Binding Sites
Sequence Homology
Amino Acid
Cell Membrane
lcsh:R
Biology and Life Sciences
Proteins
Membrane Proteins
Cell Biology
Molecular Weight
030104 developmental biology
Solubility
Biophysics
lcsh:Q
Zdroj: PLoS ONE, Vol 13, Iss 1, p e0191371 (2018)
PLoS ONE
ISSN: 1932-6203
Popis: The Nogo-B receptor (NgBR) is involved in oncogenic Ras signaling through directly binding to farnesylated Ras. It recruits farnesylated Ras to the non-lipid-raft membrane for interaction with downstream effectors. However, the cytosolic domain of NgBR itself is only partially folded. The lack of several conserved secondary structural elements makes this domain unlikely to form a complete farnesyl binding pocket. We find that inclusion of the extracellular and transmembrane domains that contain additional conserved residues to the cytosolic region results in a well folded protein with a similar size and shape to the E.coli cis-isoprenyl transferase (UPPs). Small Angle X-ray Scattering (SAXS) analysis reveals the radius of gyration (Rg) of our NgBR construct to be 18.2 A with a maximum particle dimension (Dmax) of 61.0 A. Ab initio shape modeling returns a globular molecular envelope with an estimated molecular weight of 23.0 kD closely correlated with the calculated molecular weight. Both Kratky plot and pair distribution function of NgBR scattering reveal a bell shaped peak which is characteristic of a single globularly folded protein. In addition, circular dichroism (CD) analysis reveals that our construct has the secondary structure contents similar to the UPPs. However, this result does not agree with the currently accepted topological orientation of NgBR which might partition this construct into three separate domains. This discrepancy suggests another possible NgBR topology and lends insight into a potential molecular basis of how NgBR facilitates farnesylated Ras recruitment.
Databáze: OpenAIRE
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