{"doi":"10.1016/j.jbc.2025.110923","title":"Optimized conditions for GTP loading of Ras","abstract":"Ras and the small GTPase group are essential for myriad cellular processes and cycle between GDP- and GTP-loaded states to allow stringent control of downstream signaling pathways. Biochemical studies of small GTPases can therefore require a specific nucleotide-bound state. Small GTPases possess basal intrinsic activity to process GTP into GDP plus inorganic phosphate; therefore, in vitro exchange of GDP for GTP is necessary for assays on GTP-loaded states. Here, we assess the methodology of in vitro nucleotide exchange for soluble H-Ras. We begin by describing a protocol to quantify the nucleotide-bound content of H-Ras using anion exchange chromatography and use this protocol to investigate optimal strategies for loading Ras with GTP by assessing the effects of time, temperature, H-Ras concentration, magnesium, excess nucleotide, and isoform identity. Together, we achieve a maximum of approximately 80% GTP-loading efficiency. We continue by considering storage of GTP-loaded H-Ras and present optimal conditions to minimize intrinsic GTP hydrolysis. Finally, we conclude by investigating the nucleotide composition of recombinantly expressed H-Ras encompassing cancer mutations at residues Gly12, Gly13, and Gln61 and find each to increase bound GTP compared with wildtype H-Ras. We therefore describe a methodology to quantitatively analyze the nucleotide content of small GTPases and their mutants and demonstrate conditions to achieve efficient GTP loading of Ras.","journal":"Journal of Biological Chemistry","year":2025,"id":548359,"datarank":0.10397207708399181,"base_score":0.6931471805599453,"endowment":0.6931471805599453,"self_citation_contribution":0.10397207708399181,"citation_network_contribution":0.0,"self_endowment_contribution":0.10397207708399181,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.952,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1409254,"name":"M Paul","orcid":"0000-0001-5551-1893","position":1,"is_corresponding":false},{"id":1441549,"name":"Asha P. Rollins","orcid":"0009-0004-0907-6295","position":2,"is_corresponding":false},{"id":86333,"name":"Titus J. Boggon","orcid":"0000-0003-2862-1637","position":3,"is_corresponding":false},{"id":903827,"name":"Kimberly J. Vish","orcid":"0000-0002-9394-1218","position":0,"is_corresponding":true}],"reference_count":80,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:53:58.530220Z","pmid":"41207625","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}