{"doi":"10.3390/ijms241511991","title":"Structural Characterization of Fluorescent Proteins Using Tunable Femtosecond Stimulated Raman Spectroscopy","abstract":"The versatile functions of fluorescent proteins (FPs) as fluorescence biomarkers depend on their intrinsic chromophores interacting with the protein environment. Besides X-ray crystallography, vibrational spectroscopy represents a highly valuable tool for characterizing the chromophore structure and revealing the roles of chromophore-environment interactions. In this work, we aim to benchmark the ground-state vibrational signatures of a series of FPs with emission colors spanning from green, yellow, orange, to red, as well as the solvated model chromophores for some of these FPs, using wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) in conjunction with quantum calculations. We systematically analyzed and discussed four factors underlying the vibrational properties of FP chromophores: sidechain structure, conjugation structure, chromophore conformation, and the protein environment. A prominent bond-stretching mode characteristic of the quinoidal resonance structure is found to be conserved in most FPs and model chromophores investigated, which can be used as a vibrational marker to interpret chromophore-environment interactions and structural effects on the electronic properties of the chromophore. The fundamental insights gained for these light-sensing units (e.g., protein active sites) substantiate the unique and powerful capability of wavelength-tunable FSRS in delineating FP chromophore properties with high sensitivity and resolution in solution and protein matrices. The comprehensive characterization for various FPs across a colorful palette could also serve as a solid foundation for future spectroscopic studies and the rational engineering of FPs with diverse and improved functions.","journal":"International Journal of Molecular Sciences","year":2023,"id":338645,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":17,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9603,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":820382,"name":"J. Nathan Henderson","orcid":null,"position":1,"is_corresponding":false},{"id":1071536,"name":"Dmitry A. Ruchkin","orcid":"0009-0009-8925-9388","position":2,"is_corresponding":false},{"id":1071537,"name":"Jacob M. Kirsh","orcid":"0000-0002-1444-2913","position":3,"is_corresponding":false},{"id":769346,"name":"Михаил С. Баранов","orcid":"0000-0002-9339-7603","position":4,"is_corresponding":false},{"id":1071538,"name":"Alexey M. Bogdanov","orcid":"0000-0002-5346-789X","position":5,"is_corresponding":false},{"id":704003,"name":"Jeremy H. Mills","orcid":"0000-0003-2017-1846","position":6,"is_corresponding":false},{"id":272545,"name":"Steven G. Boxer","orcid":"0000-0001-9167-4286","position":7,"is_corresponding":false},{"id":406326,"name":"Chong Fang","orcid":"0000-0002-8879-1825","position":8,"is_corresponding":false},{"id":711664,"name":"Cheng Chen","orcid":"0000-0003-0561-5226","position":0,"is_corresponding":true}],"reference_count":124,"raw_metadata":null,"created_at":"2026-07-19T01:10:40.496978Z","pmid":"37569365","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":[]}