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Characterization of two binding modes of
ANS-bacterial luciferase complex
by fluorescent spectroscopy in viscous medium
Tatyana Avsievich , Tambov State Technical University
Elena Nemtseva , Marina Gerasimova, Siberian Federal University, Krasnoyarsk
 Introduction
Intracellular media is:
• inhomogeneous,
• structured,
• has a high viscosity.
Bioluminescent reaction of bacteria –
a model process for the study of the
enzyme functioning in environments which
approximate intracellular conditions.
Fig.1 – The inhomogeneous cytoplasm of the
intact mobile cell
Dictyostelium discoideum
[Medalia O., Weber I., at al. Science, 2002]
 Introduction
Evaluation of protein binding sites under
the influence of external factors, an
important characteristic of the functioning
of enzymes in vivo and in vitro.
Objective:
to characterize influence of viscous media
on the binding characteristics of bacterial
luciferase by steady-state and timeresolved fluorescence.
Fig. 2 - Native and unfolded conformation of the
protein in the presence of osmolytes.
[C. Le Coeur at al., Life Sciences and Biology, 2005]
 Materials and methods: fluorometric titration of bacterial
luciferase by 1,8-ANS in different mediums
8-anilinonaphthalene-1-sulfonic
acid (ANS)
Upon binding to proteins:
• Quantum yield increases 2 fold
• Spectral shift 50 nm
• Electrostatic or hydrophobic interaction
• The longer fluorescence life time (>15 ns) has
been attributed to the internal binding sites
and the shorter (<10 ns) to the external sites
Bacterial luciferase
Photobacterium leiognathi (L)
Buffer 0,05 M
(η=1,002 сP)
Glycerol, 40 wt%,
(η=3,75 сP)
Features:
• Substrates: long chain aldehyde and
reduced flavin mononucleotide
• Heterodimer, ~80 kDa
• The exact number and affinities of its
binding sites have not been determined yet
Sucrose, 40 wt%,
(η=6,17 сP)
 Materials and methods: fluorometric titration of bacterial
luciferase by 1,8-ANS in mediums of different nature
Buffer 0,05 M
(η=1,002 cP)
The chosen viscous media have the same effect
decrease bioluminescence in vitro by more than 2fold compared with the control (buffer 0,05 M)
Glycerol, 40 wt%,
(η=3,75 cP)
Sucrose, 40 wt%,
(η=6,17 cP)
 Methods: Fluorescence spectroscopy
Spectrofluorimeter Fluorolog-3-22
(Horiba Jobin Yvon, France)
Steady-state fluorescence
Time-resolved fluorescence
Correction of fluorescence
intensities for inner filter effect:
I
corr
 I obs 10
(
D360 D470)
2
,
I corr – the true intensity of the fluorescence, I obs experimentally measured intensity, D360, D470 –
absorption at wavelengths of 360 and 470 nm,
respectively
The lifetimes were found from the
dependence of intensity on time :
I t   i exp  t  i ,
i and i – amplitudes and the
lifetimes of the i-components
 Results: 1,8-ANS fluorescence in the presence of luciferase
(fluorescence lifetime)
Table 1. - Lifetime components for fluorescence of [ANS+luciferase] (1 , 2 , 3):
Media
1 , ns
2, ns
3, ns
Buffer 0,05 М
0,6±0,06
5,78±0,06
14,7±1,4
Glycerol, 40%
0,6±0,013
4,77±0,12
12,3±0,08
Sucrose, 40%
0,44±0,01
4,52±0,09
12,4±0,08
Free ANS
ANS, bound with external sites
ANS, bound with internal sites
Fig.3 - fluorescence decays for ANS in the presence of
bacterial luciferase in different mediums
 Results: Deconvolution of steady-state fluorescence titration
curve (Iss) into lifetime components 2 and 3
Nonlinear fitting of the titration curves:
where y – experimental fluorescence, F is a fluorescence scaling factor Kd - dissociation constant, P - total protein concentration
(luciferase), Lt - the total ligand concentration (ANS), n – number of binding sites.
Fig. 4 - Deconvolution of ANS fluorescence titration curves at 430 nm into lifetime components
(markers) and their nonlinear fitting (straight lines).
Iss – overall intensity of the fluorescence of the probe in the steady-state excitation
Issf2 – fluorescence intensity of external binding sites (2 =4-6 ns)
Issf3 –fluorescence intensity of internal binding sites (3 = 12-14 ns)
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
Results: influence of viscous media on the binding
characteristics of the protein
Table 2. – Characteristics of 1,8-ANS binding to the bacterial luciferase
Media
Viscosity, cP
2 -external sites
3 -internal sites
Kd1, µM
n1
Kd2, µM
n2
Buffer 0,05 М
1,002
8,7±4,5
16,1±1,8
1,3±0,3
2,15±0,19
Glycerol 40%
3,75
84±23,8
3,8±2,9
14,2±2,5
1,22±0,68
Sucrose 40%
6,17
44±8,9
1,4±0,7
6,4±2,4
3,2±1,26
o In viscous media the weakening of the binding of the probe with internal and
external sites was obtained. This effect is stronger in glycerol than in sucrose.
o The number of external binding sites dramatically reduced in viscous media.
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 Discussion: mechanisms of ANS interaction with luciferase
o Snp emits when ANS is fixed in non-planar
conformation (bound to the protein)
o SCT emits when ANS is in planar
conformation
o SCT is quenched by water molecules
o (+)-charged amino acids play role in the
interaction with proteins.
460-480 nm
510-540 nm
Fig. 5 – The model of two emmiting states of 1,8-ANS
[Gufeng at al., Biochimica et Biophysica Acta., 2006]
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 Discussion: mechanisms of ANS interaction with luciferase
o Luciferase’s active site contains charged amino
acids and a hydrophobic pocket;
o ANS competes with the FMH to the binding
with luciferase  possibly ANS binds to the
active site of the protein
Adding of glycerin or sucrose causes:
 The decreasing of the dielectric constant of
the medium  enhancement of electrostatic
interactions increase in rigidity of structure
protein  difficulties entering ligands in the
internal sites
or
Fig. 4 – CPK representation of bacterial luciferase
molecule (PDB id: 3FGC): hydrophobic (green), positively
charged (indigo) and other (white) amino acid residuals,
FMN in active center is red.
 Preferential hydration of proteins  excess
water leads to the quenching of the probe 
the surface charge of the luciferase is changed
 no binding
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 Conclusions:
1)
From interaction of 1,8-ANS with bacterial luciferase in all investigated media two
types of fluorophores with short (5 ns) and long (12-15 ns) fluorescence lifetime are
formed corresponding to the binding of the probe with the internal and external sites
of the protein molecule.
2)
Binding of 1,8-ANS to internal sites of the bacterial luciferase is characterized by a
higher affinity (Kd = 1,3 ± 0,9 μM) than with external (Kd = 8,7 ± 0,5 μM).
3)
In viscous media with glycerol and sucrose the number of external binding sites of
the bacterial luciferase is significantly reduced, the interaction of the probe with all
centers weakens.
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