David P. Broughton, Chloe G. Holod, Angelica Camilo-Contreras, Darcy R. Harris, Scott H. Brewer and Christine M. Phillips-Piro
{"title":"调节绿色荧光蛋白† 与 pH 值相关的光物理特性","authors":"David P. Broughton, Chloe G. Holod, Angelica Camilo-Contreras, Darcy R. Harris, Scott H. Brewer and Christine M. Phillips-Piro","doi":"10.1039/D4RA05058D","DOIUrl":null,"url":null,"abstract":"<p >The photophysical properties of the β-barrel superfolder green fluorescent protein (sfGFP) arise from the chromophore that forms post-translationally in the interior of the protein. Specifically, the protonation state of the side chain of tyrosine 66 in the chromophore, in addition to the network of hydrogen bonds between the chromophore and surrounding residues, is directly related to the electronic absorbance and emission properties of the protein. The pH dependence of the photophysical properties of this protein were modulated by the genetic, site-specific incorporation of 3-nitro-<small>L</small>-tyrosine (mNO<small><sub>2</sub></small>Y) at site 66 in sfGFP. The altered photophysical properties of this noncanonical amino acid (ncAA) sfGFP construct were assessed by absorbance and fluorescence spectroscopies. Notably, a comparison of the p<em>K</em><small><sub>a</sub></small> of the 3-nitrophenol side chain of mNO<small><sub>2</sub></small>Y incorporated in the protein relative to the phenol side chain of the tyrosine at site 66 in the native chromophore as well as the p<em>K</em><small><sub>a</sub></small> of the 3-nitrophenol side chain of the free ncAA were measured and are compared. A structural analysis of the ncAA containing sfGFP construct is presented to yield molecular insight into the origin of the altered absorbance and fluorescence properties of the protein.</p>","PeriodicalId":102,"journal":{"name":"RSC Advances","volume":null,"pages":null},"PeriodicalIF":3.9000,"publicationDate":"2024-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/ra/d4ra05058d?page=search","citationCount":"0","resultStr":"{\"title\":\"Modulating the pH dependent photophysical properties of green fluorescent protein†\",\"authors\":\"David P. Broughton, Chloe G. Holod, Angelica Camilo-Contreras, Darcy R. Harris, Scott H. Brewer and Christine M. Phillips-Piro\",\"doi\":\"10.1039/D4RA05058D\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p >The photophysical properties of the β-barrel superfolder green fluorescent protein (sfGFP) arise from the chromophore that forms post-translationally in the interior of the protein. Specifically, the protonation state of the side chain of tyrosine 66 in the chromophore, in addition to the network of hydrogen bonds between the chromophore and surrounding residues, is directly related to the electronic absorbance and emission properties of the protein. The pH dependence of the photophysical properties of this protein were modulated by the genetic, site-specific incorporation of 3-nitro-<small>L</small>-tyrosine (mNO<small><sub>2</sub></small>Y) at site 66 in sfGFP. The altered photophysical properties of this noncanonical amino acid (ncAA) sfGFP construct were assessed by absorbance and fluorescence spectroscopies. Notably, a comparison of the p<em>K</em><small><sub>a</sub></small> of the 3-nitrophenol side chain of mNO<small><sub>2</sub></small>Y incorporated in the protein relative to the phenol side chain of the tyrosine at site 66 in the native chromophore as well as the p<em>K</em><small><sub>a</sub></small> of the 3-nitrophenol side chain of the free ncAA were measured and are compared. A structural analysis of the ncAA containing sfGFP construct is presented to yield molecular insight into the origin of the altered absorbance and fluorescence properties of the protein.</p>\",\"PeriodicalId\":102,\"journal\":{\"name\":\"RSC Advances\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.9000,\"publicationDate\":\"2024-10-17\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://pubs.rsc.org/en/content/articlepdf/2024/ra/d4ra05058d?page=search\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"RSC Advances\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://pubs.rsc.org/en/content/articlelanding/2024/ra/d4ra05058d\",\"RegionNum\":3,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"CHEMISTRY, MULTIDISCIPLINARY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"RSC Advances","FirstCategoryId":"92","ListUrlMain":"https://pubs.rsc.org/en/content/articlelanding/2024/ra/d4ra05058d","RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"CHEMISTRY, MULTIDISCIPLINARY","Score":null,"Total":0}
Modulating the pH dependent photophysical properties of green fluorescent protein†
The photophysical properties of the β-barrel superfolder green fluorescent protein (sfGFP) arise from the chromophore that forms post-translationally in the interior of the protein. Specifically, the protonation state of the side chain of tyrosine 66 in the chromophore, in addition to the network of hydrogen bonds between the chromophore and surrounding residues, is directly related to the electronic absorbance and emission properties of the protein. The pH dependence of the photophysical properties of this protein were modulated by the genetic, site-specific incorporation of 3-nitro-L-tyrosine (mNO2Y) at site 66 in sfGFP. The altered photophysical properties of this noncanonical amino acid (ncAA) sfGFP construct were assessed by absorbance and fluorescence spectroscopies. Notably, a comparison of the pKa of the 3-nitrophenol side chain of mNO2Y incorporated in the protein relative to the phenol side chain of the tyrosine at site 66 in the native chromophore as well as the pKa of the 3-nitrophenol side chain of the free ncAA were measured and are compared. A structural analysis of the ncAA containing sfGFP construct is presented to yield molecular insight into the origin of the altered absorbance and fluorescence properties of the protein.
期刊介绍:
An international, peer-reviewed journal covering all of the chemical sciences, including multidisciplinary and emerging areas. RSC Advances is a gold open access journal allowing researchers free access to research articles, and offering an affordable open access publishing option for authors around the world.