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Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein

View Article: PubMed Central - PubMed

ABSTRACT

Fluorescence approaches have been widely used for elucidating the dynamics of protein-membrane interactions in cells and model systems. However, non-specific multi-site fluorescent labeling often results in a loss of native structure and function, and single cysteine labeling is not feasible when native cysteines are required to support a protein’s folding or catalytic activity. Here, we develop a method using genetic incorporation of non-natural amino acids and bio-orthogonal chemistry to site-specifically label with a single fluorescent small molecule or protein the myristoyl-switch protein recoverin, which is involved in rhodopsin-mediated signaling in mammalian visual sensory neurons. We demonstrate reversible Ca2+-responsive translocation of labeled recoverin to membranes and show that recoverin favors membranes with negative curvature and high lipid fluidity in complex heterogeneous membranes, which confers spatio-temporal control over down-stream signaling events. The site-specific orthogonal labeling technique is promising for structural, dynamical, and functional studies of many lipid-anchored membrane protein switches.

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Genetic incorporation of the non-natural amino acid AZF into two selected positions of recoverin.(a) Three dimensional structures of recoverin in its Ca2+-free (PDB code: 1IKU; left) and Ca2+-bound (PDB code: 1JSA; right) forms. Sites selected for mutation are marked with arrows. The N-myristoyl chain is represented by red spheres. (b) SDS-PAGE of purified recoverin and variants: Lane 1, non-myristoylated Rec-F23AZF; Lane 2, myristoylated recoverin; Lane 3, mRec-F23AZF; Lane 4, mRec-F158AZF. (c) Far-UV CD spectra of recoverin and variants in the absence or presence of 1 mM Ca2+. (d) In-gel fluorescence of recoverin and variants reacted with DBCO-PEG4-carboxyrhodamine (left) or DBCO-NBD (right): Lane 1, mRec; Lane 2, mRec-F23AZF; Lane 3, mRec-F158AZF. The labeled protein bands run at 24 kDa as shown with the mCherry reference in Supplementary Fig. 4.
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f1: Genetic incorporation of the non-natural amino acid AZF into two selected positions of recoverin.(a) Three dimensional structures of recoverin in its Ca2+-free (PDB code: 1IKU; left) and Ca2+-bound (PDB code: 1JSA; right) forms. Sites selected for mutation are marked with arrows. The N-myristoyl chain is represented by red spheres. (b) SDS-PAGE of purified recoverin and variants: Lane 1, non-myristoylated Rec-F23AZF; Lane 2, myristoylated recoverin; Lane 3, mRec-F23AZF; Lane 4, mRec-F158AZF. (c) Far-UV CD spectra of recoverin and variants in the absence or presence of 1 mM Ca2+. (d) In-gel fluorescence of recoverin and variants reacted with DBCO-PEG4-carboxyrhodamine (left) or DBCO-NBD (right): Lane 1, mRec; Lane 2, mRec-F23AZF; Lane 3, mRec-F158AZF. The labeled protein bands run at 24 kDa as shown with the mCherry reference in Supplementary Fig. 4.

Mentions: Recoverin undergoes a drastic conformational change upon binding of two Ca2+ ions at EF-hands 2 and 3, leading to the extrusion of an N-terminal myristoyl group and exposure of a hydrophobic groove6. To minimize perturbation of this important functional switch, potential sites for non-natural amino acid (NNAA) incorporation were examined in the context of physicochemical and functional constraints. The NNAA AZF is a phenylalanine analog with an azide substituted in para position. Since AZF has a similar chemical structure and hydrophobicity as phenylalanine and tyrosine, we considered all fourteen phenylalanines and five tyrosines of the native amino acid sequence of recoverin as potential candidates for AZF substitution. It has been reported that the efficiency of dye labeling largely depends on the solvent accessibility of targeted amino acid residues22. As assessed by the ASA-View Calculator23, each candidate exhibited varying degrees of the relative solvent accessibility depending on the position and the Ca2+-induced conformational change (Supplementary Fig. 1). Two phenylalanines in positions 23 (F23 in EF-1) and 158 (F158 in EF-4) were selected for mutation since they have both relatively high solvent accessibilities in the Ca2+-free and Ca2+-bound states, and neither of them is directly involved in the membrane-binding (Fig. 1a).


Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein
Genetic incorporation of the non-natural amino acid AZF into two selected positions of recoverin.(a) Three dimensional structures of recoverin in its Ca2+-free (PDB code: 1IKU; left) and Ca2+-bound (PDB code: 1JSA; right) forms. Sites selected for mutation are marked with arrows. The N-myristoyl chain is represented by red spheres. (b) SDS-PAGE of purified recoverin and variants: Lane 1, non-myristoylated Rec-F23AZF; Lane 2, myristoylated recoverin; Lane 3, mRec-F23AZF; Lane 4, mRec-F158AZF. (c) Far-UV CD spectra of recoverin and variants in the absence or presence of 1 mM Ca2+. (d) In-gel fluorescence of recoverin and variants reacted with DBCO-PEG4-carboxyrhodamine (left) or DBCO-NBD (right): Lane 1, mRec; Lane 2, mRec-F23AZF; Lane 3, mRec-F158AZF. The labeled protein bands run at 24 kDa as shown with the mCherry reference in Supplementary Fig. 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5015116&req=5

f1: Genetic incorporation of the non-natural amino acid AZF into two selected positions of recoverin.(a) Three dimensional structures of recoverin in its Ca2+-free (PDB code: 1IKU; left) and Ca2+-bound (PDB code: 1JSA; right) forms. Sites selected for mutation are marked with arrows. The N-myristoyl chain is represented by red spheres. (b) SDS-PAGE of purified recoverin and variants: Lane 1, non-myristoylated Rec-F23AZF; Lane 2, myristoylated recoverin; Lane 3, mRec-F23AZF; Lane 4, mRec-F158AZF. (c) Far-UV CD spectra of recoverin and variants in the absence or presence of 1 mM Ca2+. (d) In-gel fluorescence of recoverin and variants reacted with DBCO-PEG4-carboxyrhodamine (left) or DBCO-NBD (right): Lane 1, mRec; Lane 2, mRec-F23AZF; Lane 3, mRec-F158AZF. The labeled protein bands run at 24 kDa as shown with the mCherry reference in Supplementary Fig. 4.
Mentions: Recoverin undergoes a drastic conformational change upon binding of two Ca2+ ions at EF-hands 2 and 3, leading to the extrusion of an N-terminal myristoyl group and exposure of a hydrophobic groove6. To minimize perturbation of this important functional switch, potential sites for non-natural amino acid (NNAA) incorporation were examined in the context of physicochemical and functional constraints. The NNAA AZF is a phenylalanine analog with an azide substituted in para position. Since AZF has a similar chemical structure and hydrophobicity as phenylalanine and tyrosine, we considered all fourteen phenylalanines and five tyrosines of the native amino acid sequence of recoverin as potential candidates for AZF substitution. It has been reported that the efficiency of dye labeling largely depends on the solvent accessibility of targeted amino acid residues22. As assessed by the ASA-View Calculator23, each candidate exhibited varying degrees of the relative solvent accessibility depending on the position and the Ca2+-induced conformational change (Supplementary Fig. 1). Two phenylalanines in positions 23 (F23 in EF-1) and 158 (F158 in EF-4) were selected for mutation since they have both relatively high solvent accessibilities in the Ca2+-free and Ca2+-bound states, and neither of them is directly involved in the membrane-binding (Fig. 1a).

View Article: PubMed Central - PubMed

ABSTRACT

Fluorescence approaches have been widely used for elucidating the dynamics of protein-membrane interactions in cells and model systems. However, non-specific multi-site fluorescent labeling often results in a loss of native structure and function, and single cysteine labeling is not feasible when native cysteines are required to support a protein’s folding or catalytic activity. Here, we develop a method using genetic incorporation of non-natural amino acids and bio-orthogonal chemistry to site-specifically label with a single fluorescent small molecule or protein the myristoyl-switch protein recoverin, which is involved in rhodopsin-mediated signaling in mammalian visual sensory neurons. We demonstrate reversible Ca2+-responsive translocation of labeled recoverin to membranes and show that recoverin favors membranes with negative curvature and high lipid fluidity in complex heterogeneous membranes, which confers spatio-temporal control over down-stream signaling events. The site-specific orthogonal labeling technique is promising for structural, dynamical, and functional studies of many lipid-anchored membrane protein switches.

No MeSH data available.


Related in: MedlinePlus