4O8G

Structure of Infrared Fluorescent Protein 1.4

PDB DOI: https://doi.org/10.2210/pdb4O8G/pdb

Classification: FLUORESCENT PROTEIN
Organism(s): Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539
Expression System: Escherichia coli BL21
Mutation(s): Yes

Deposited: 2013-12-27 Released: 2014-10-01
Deposition Author(s): Bhattacharya, S., Forest, K.T.

Experimental Data Snapshot

Method: X-RAY DIFFRACTION
Resolution: 1.65 Å
R-Value Free: 0.211
R-Value Work: 0.185
R-Value Observed: 0.186

wwPDB Validation 3D Report Full Report

Ligand Structure Quality Assessment

This is version 1.3 of the entry. See complete history.

Literature

Origins of fluorescence in evolved bacteriophytochromes.

Bhattacharya, S., Auldridge, M.E., Lehtivuori, H., Ihalainen, J.A., Forest, K.T.

(2014) J Biol Chem 289: 32144-32152

PubMed: 25253687 Search on PubMedSearch on PubMed Central
DOI: https://doi.org/10.1074/jbc.M114.589739
Primary Citation of Related Structures:
4IJG, 4O8G

PubMed Abstract:
Use of fluorescent proteins to study in vivo processes in mammals requires near-infrared (NIR) biomarkers that exploit the ability of light in this range to penetrate tissue. Bacteriophytochromes (BphPs) are photoreceptors that couple absorbance of NIR light to photoisomerization, protein conformational changes, and signal transduction. BphPs have been engineered to form NIR fluorophores, including IFP1.4, Wi-Phy, and the iRFP series, initially by replacement of Asp-207 by His. This position was suggestive because its main chain carbonyl is within hydrogen-bonding distance to pyrrole ring nitrogens of the biliverdin chromophore, thus potentially functioning as a crucial transient proton sink during photoconversion. To explain the origin of fluorescence in these phytofluors, we solved the crystal structures of IFP1.4 and a comparison non-fluorescent monomeric phytochrome DrCBDmon. Met-186 and Val-288 in IFP1.4 are responsible for the formation of a tightly packed hydrophobic hub around the biliverdin D ring. Met-186 is also largely responsible for the blue-shifted IFP1.4 excitation maximum relative to the parent BphP. The structure of IFP1.4 revealed decreased structural heterogeneity and a contraction of two surface regions as direct consequences of side chain substitutions. Unexpectedly, IFP1.4 with Asp-207 reinstalled (IFPrev) has a higher fluorescence quantum yield (∼9%) than most NIR phytofluors published to date. In agreement, fluorescence lifetime measurements confirm the exceptionally long excited state lifetimes, up to 815 ps, in IFP1.4 and IFPrev. Our research helps delineate the origin of fluorescence in engineered BphPs and will facilitate the wide-spread adoption of phytofluors as biomarkers.

Organizational Affiliation

Departments of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706 and.

Macromolecule Content

Total Structure Weight: 37.06 kDa
Atom Count: 2,745
Modelled Residue Count: 311
Deposited Residue Count: 329
Unique protein chains: 1

Macromolecules

Find similar proteins by:

(by identity cutoff) | 3D Structure

Entity ID: 1
Molecule	Chains	Sequence Length	Organism	Details	Image
Bacteriophytochrome	A	329	Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539	Mutation(s): 12 Gene Names: bphP, DR_A0050
UniProt
Find proteins for Q9RZA4 (Deinococcus radiodurans (strain ATCC 13939 / DSM 20539 / JCM 16871 / CCUG 27074 / LMG 4051 / NBRC 15346 / NCIMB 9279 / VKM B-1422 / R1)) Explore Q9RZA4 Go to UniProtKB: Q9RZA4
Entity Groups
Sequence Clusters	30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt Group	Q9RZA4
Sequence Annotations Expand
Reference Sequence

Small Molecules

Ligands 2 Unique
ID	Chains	Name / Formula / InChI Key	2D Diagram	3D Interactions
LBW Query on LBW Download Ideal Coordinates CCD File SDF format, chain C [auth A] MOL2 format, chain C [auth A]	C [auth A]	3-[2-[(Z)-[5-[(Z)-(4-ethenyl-3-methyl-5-oxidanylidene-pyrrol-2-ylidene)methyl]-3-(3-hydroxy-3-oxopropyl)-4-methyl-pyrrol-1-ium-2-ylidene]methyl]-5-[(Z)-[(3E,4R)-3-ethylidene-4-methyl-5-oxidanylidene-pyrrolidin-2-ylidene]methyl]-4-methyl-1H-pyrrol-3-yl]propanoic acid C₃₃ H₃₇ N₄ O₆ DKMLMZVDTGOEGU-UAWLBFNISA-O		Interactions Focus chain C [auth A] Interactions & Density Focus chain C [auth A]
LBV Query on LBV Download Ideal Coordinates CCD File SDF format, chain B [auth A] MOL2 format, chain B [auth A]	B [auth A]	3-[2-[(Z)-[3-(2-carboxyethyl)-5-[(Z)-(4-ethenyl-3-methyl-5-oxidanylidene-pyrrol-2-ylidene)methyl]-4-methyl-pyrrol-1-ium -2-ylidene]methyl]-5-[(Z)-[(3E)-3-ethylidene-4-methyl-5-oxidanylidene-pyrrolidin-2-ylidene]methyl]-4-methyl-1H-pyrrol-3- yl]propanoic acid C₃₃ H₃₇ N₄ O₆ DKMLMZVDTGOEGU-ISEYCTJISA-O		Interactions Focus chain B [auth A] Interactions & Density Focus chain B [auth A]

Experimental Data & Validation

Experimental Data

Method: X-RAY DIFFRACTION
Resolution: 1.65 Å
R-Value Free: 0.211
R-Value Work: 0.185
R-Value Observed: 0.186
Space Group: C 1 2 1

Unit Cell:

Length ( Å )	Angle ( ˚ )
a = 96.162	α = 90
b = 53.228	β = 90.6
c = 66.796	γ = 90

Software Package:

Software Name	Purpose
HKL-2000	data collection
PHASER	phasing
REFMAC	refinement
HKL-2000	data reduction
HKL-2000	data scaling

Structure Validation

View Full Validation Report

Ligand Structure Quality Assessment

Entry History

Deposition Data

Released Date: 2014-10-01

Deposition Author(s):

Bhattacharya, S.

Forest, K.T.

Revision History (Full details and data files)

Version 1.0: 2014-10-01
Type: Initial release
Version 1.1: 2014-10-08
Changes: Database references
Version 1.2: 2014-12-03
Changes: Database references
Version 1.3: 2023-09-20
Changes: Data collection, Database references, Derived calculations, Refinement description, Structure summary