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Related Concept Videos

Small GTPases - Ras and Rho01:24

Small GTPases - Ras and Rho

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Ras and Rho are small monomeric GTPases that act downstream of receptor tyrosine kinase (RTK) and regulate various cellular processes. These GTPases switch between active and inactive states by binding to guanine nucleotides.
Three regulatory proteins control their activity:
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The Ras Gene02:38

The Ras Gene

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The Ras-gene-encoded proteins are regulators of signaling pathways controlling cell proliferation, differentiation, or cell survival. The Ras-gene family in humans constitutes three primary members—the HRas, NRas, and KRas. These genes code for four functionally distinct yet closely related proteins—the HRas, NRas, KRas4A, and KRas4B. The involvement of mutant Ras genes in human cancer was first discovered in 1982 and is among the most common causes of human tumorigenesis.
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GTPases and their Regulation02:14

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Guanine nucleotide-binding proteins (G-proteins), also known as GTPases, are a superfamily of proteins that regulate many cellular processes, such as cell signaling, vesicular transport, and the regulation of cell shape and motility. Mutation or dysfunction of these proteins can lead to disease. There are around 40,000 known G-proteins that can broadly be classified into two groups ‒  small G-proteins consisting of a single domain and large multi-domain G-proteins.
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Rab Proteins01:14

Rab Proteins

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Rab proteins constitute the largest family of monomeric GTPases, of which 70 members are present in humans. Rab proteins and their effectors regulate consecutive stages of vesicle transport such as vesicle transport, docking, and fusion to the correct recipient membrane.
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Rab Cascades01:25

Rab Cascades

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Rab GTPases act in a regulated cascade during membrane fusion, helping the lipid bilayers mix. The Rab family of proteins are active when bound to GTP, and inactive when bound to GDP. Hence, they act as guanine nucleotide-dependent molecular switches. Rab-GTP recognizes and binds to long or short-range tethering proteins to capture the target vesicle. These tethers coordinate with SNAREs on the vesicle and the target membrane to assemble the trans SNARE complex that locks the mixing bilayers.
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Related Experiment Video

Updated: Jan 11, 2026

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay
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Optimized conditions for GTP loading of Ras.

Kimberly J Vish1, Maxum E Paul1, Asha P Rollins2

  • 1Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.

The Journal of Biological Chemistry
|November 9, 2025
PubMed
Summary

Researchers optimized methods for loading Ras proteins with GTP, achieving up to 80% efficiency. This work improves the study of Ras signaling pathways and cancer mutations by ensuring accurate nucleotide-bound states for H-Ras (Human Ras GTPase).

Keywords:
GDPGTPGTPase cyclingRassmall GTPase

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cellular Signaling

Background:

  • Ras proteins and small GTPases are crucial for cellular functions, cycling between GDP- and GTP-bound states.
  • Accurate nucleotide-bound states are essential for studying small GTPase signaling pathways in vitro.
  • Small GTPases exhibit intrinsic GTPase activity, necessitating in vitro nucleotide exchange for GTP-bound state assays.

Purpose of the Study:

  • To optimize and validate a methodology for in vitro nucleotide exchange in soluble H-Ras.
  • To determine optimal conditions for efficient GTP loading and storage of GTP-loaded H-Ras.
  • To analyze the nucleotide composition of H-Ras mutants associated with cancer.

Main Methods:

  • Quantification of nucleotide-bound H-Ras using anion exchange chromatography.
  • Systematic assessment of factors influencing GTP loading: time, temperature, protein concentration, magnesium, and nucleotide excess.
  • Evaluation of storage conditions to minimize GTP hydrolysis.
  • Analysis of nucleotide content in H-Ras mutants (G12, G13, Q61).

Main Results:

  • Developed a protocol for quantifying nucleotide-bound H-Ras.
  • Achieved a maximum of approximately 80% GTP-loading efficiency under optimized conditions.
  • Identified optimal storage conditions to minimize intrinsic GTP hydrolysis.
  • Found that cancer-associated H-Ras mutants (G12, G13, Q61) exhibit increased bound GTP compared to wild-type.

Conclusions:

  • Established a robust methodology for quantitative analysis of small GTPase nucleotide content.
  • Demonstrated conditions for efficient GTP loading of Ras proteins, crucial for biochemical studies.
  • Highlighted differences in nucleotide binding for cancer-associated Ras mutants, providing insights into their altered function.