1F0B

CRYSTAL STRUCTURE OF THE GREEN FLUORESCENT PROTEIN (GFP) VARIANT YFP-H148Q


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.10 Å
  • R-Value Work: 0.188 
  • R-Value Observed: 0.188 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Crystallographic and energetic analysis of binding of selected anions to the yellow variants of green fluorescent protein.

Wachter, R.M.Yarbrough, D.Kallio, K.Remington, S.J.

(2000) J Mol Biol 301: 157-171

  • DOI: https://doi.org/10.1006/jmbi.2000.3905
  • Primary Citation of Related Structures:  
    1F09, 1F0B

  • PubMed Abstract: 

    The fluorescence emission of yellow fluorescent proteins (YFPs) has been shown to respond rapidly and reversibly to changes in the concentration of some small anions such as halides; this allows for the use of YFPs as genetically encodable Cl(-) sensors that may be targeted to specific organelles in living cells. Fluorescence is suppressed due to protonation of the chromophore upon anion binding, with a stronger level of interaction at low pH values. At pH 6.0, the apparent dissociation constant (K(app)) for Cl(-) is 32 mM for YFP and 22 mM for YFP-H148Q, whereas at pH 7.5, K(app) is 777 mM and 154 mM, respectively. In the cytosol, YFP-H148Q appears most promising as a halide sensor due to its high degree of sensitivity towards I(-) (K(app)=23 mM at pH 7.5). To aid in the design of variants with improved levels of specificity and affinity for Cl(-), we solved apo and I(-)-bound crystal structures of YFP-H148Q to 2.1 A resolution. The halide-binding site is found near van der Waals contact with the chromophore imidazolinone oxygen atom, in a small buried cavity adjacent to Arg96, which provides electrostatic stabilization. The halide ion is hydrogen bonded to the phenol group of T203Y, consistent with a mutational analysis that indicates that T203Y is indispensible for tight binding. A series of conformational changes occurs in the amphiphilic site upon anion binding, which appear to be propagated to the beta-bulge region around residue 148 on the protein surface. Anion binding raises the chromophore pK(a) values, since delocalization of the phenolate negative charge over the chromophore skeleton is suppressed. Extraction of microscopic binding constants for the linked equilibrium between anion and proton binding indicates that anion selectivity by YFP is related to hydration forces. Specific suggestions to improve Cl(-) binding to YFP-H148Q based on size and hydration energy are proposed.


  • Organizational Affiliation

    Institute of Molecular Biology Departments of Physics and Biology, University of Oregon, Eugene, OR, 97403, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
GREEN FLUORESCENT PROTEIN236Aequorea victoriaMutation(s): 6 
UniProt
Find proteins for P42212 (Aequorea victoria)
Explore P42212 
Go to UniProtKB:  P42212
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP42212
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Modified Residues  1 Unique
IDChains TypeFormula2D DiagramParent
CR2
Query on CR2
A
L-PEPTIDE LINKINGC13 H13 N3 O4GLY, TYR, GLY
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.10 Å
  • R-Value Work: 0.188 
  • R-Value Observed: 0.188 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 51.661α = 90
b = 62.646β = 90
c = 66.183γ = 90
Software Package:
Software NamePurpose
TNTrefinement
DENZOdata reduction
SCALEPACKdata scaling
TNTphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2000-11-17
    Type: Initial release
  • Version 1.1: 2008-04-27
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2021-11-03
    Changes: Database references, Derived calculations
  • Version 1.4: 2024-10-30
    Changes: Data collection, Structure summary