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While the microsecond collapse of horse cyt c has been intensely studied, its molecular mechanism remains elusive. To characterize the structural change, we prepared three cyt c variants labeled with a fluorescent dye at distinct sites (D50, A83, and E104). We track their dynamics via FRET between the dye and the heme by two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS), which has single-molecule sensitivity and sub-microsecond time resolution. The results reveal that the collapse exhibits two-state dynamics between structurally distributed ensembles, occurring on a time scale of tens of microseconds across all variants. This indicates a global contraction of the whole protein structure with a substantial free energy barrier. A slight delay observed for the D50 variant suggests the late folding of its associated loop. This study demonstrates the capability of 2D FLCS to characterize a small free-energy barrier in complex biophysical processes.<\/jats:p>","DOI":"10.1021\/acs.jpclett.6c01834","type":"journal-article","created":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T18:02:10Z","timestamp":1788976930000},"page":"10803-10810","source":"Crossref","is-referenced-by-count":0,"title":["Microsecond Barrier-Crossing Collapse in Cytochrome\n                    <i>c<\/i>\n                    Folding Revealed by Two-Dimensional Fluorescence Lifetime\nCorrelation Spectroscopy"],"prefix":"10.1021","volume":"17","author":[{"given":"Miyuki","family":"Sakaguchi","sequence":"first","affiliation":[{"name":"RIKEN , , 2-1 Hirosawa , ,","place":["Wako, Japan, 351-0198"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masaru","family":"Yamanaka","sequence":"additional","affiliation":[{"name":"Nara Institute of Science and Technology , , 8916-5 Takayama-cho , ,","place":["Ikoma, Japan, 630-0192"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3227-8376","authenticated-orcid":true,"given":"Shun","family":"Hirota","sequence":"additional","affiliation":[{"name":"Nara Institute of Science and Technology , , 8916-5 Takayama-cho , ,","place":["Ikoma, Japan, 630-0192"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5714-5997","authenticated-orcid":true,"given":"Kunihiko","family":"Ishii","sequence":"additional","affiliation":[{"name":"RIKEN , , 2-1 Hirosawa , ,","place":["Wako, Japan, 351-0198"]},{"name":"RIKEN Center for Advanced Photonics (RAP), RIKEN , , 2-1 Hirosawa , ,","place":["Wako, Japan, 351-0198"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6340-8535","authenticated-orcid":true,"given":"Tahei","family":"Tahara","sequence":"additional","affiliation":[{"name":"RIKEN , , 2-1 Hirosawa , ,","place":["Wako, Japan, 351-0198"]},{"name":"RIKEN Center for Advanced Photonics (RAP), RIKEN , , 2-1 Hirosawa , ,","place":["Wako, Japan, 351-0198"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,9]]},"reference":[{"key":"2026092408415846200_cit1","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.sbi.2015.11.006","article-title":"Where the complex things are: single\nmolecule and ensemble spectroscopic investigations of protein folding\ndynamics","volume":"36","author":"Takahashi","year":"2016","journal-title":"Curr. 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