Tatyana Avsievich, Tambov State Technical University Elena Nemtseva, Marina Gerasimova, Siberian...

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Tatyana Avsievich , Tambov State Technical University Elena Nemtseva , Marina Gerasimova, Siberian Federal University, Krasnoyarsk Characterization of two binding modes of ANS-bacterial luciferase complex by fluorescent spectroscopy in viscous medium

Transcript of Tatyana Avsievich, Tambov State Technical University Elena Nemtseva, Marina Gerasimova, Siberian...

Page 1: Tatyana Avsievich, Tambov State Technical University Elena Nemtseva, Marina Gerasimova, Siberian Federal University, Krasnoyarsk Characterization of two.

Tatyana Avsievich , Tambov State Technical UniversityElena Nemtseva , Marina Gerasimova, Siberian Federal University, Krasnoyarsk

Characterization of two binding modes of ANS-bacterial luciferase complex

by fluorescent spectroscopy in viscous medium

Page 2: Tatyana Avsievich, Tambov State Technical University Elena Nemtseva, Marina Gerasimova, Siberian Federal University, Krasnoyarsk Characterization of two.

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]

Page 3: Tatyana Avsievich, Tambov State Technical University Elena Nemtseva, Marina Gerasimova, Siberian Federal University, Krasnoyarsk Characterization of two.

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 time-resolved 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]

Page 4: Tatyana Avsievich, Tambov State Technical University Elena Nemtseva, Marina Gerasimova, Siberian Federal University, Krasnoyarsk Characterization of two.

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

8-anilinonaphthalene-1-sulfonic acid (ANS)

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

Bacterial luciferase Photobacterium leiognathi (L)

Materials and methods: fluorometric titration of bacterial luciferase by 1,8-ANS in different mediums

Buffer 0,05 M (η=1,002 сP)

Glycerol, 40 wt%, (η=3,75 сP)

Sucrose, 40 wt%, (η=6,17 сP)

Page 5: Tatyana Avsievich, Tambov State Technical University Elena Nemtseva, Marina Gerasimova, Siberian Federal University, Krasnoyarsk Characterization of two.

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)

Glycerol, 40 wt%, (η=3,75 cP)

Sucrose, 40 wt%, (η=6,17 cP)

The chosen viscous media have the same effectdecrease bioluminescence in vitro by more than 2-fold compared with the control (buffer 0,05 M)

Page 6: Tatyana Avsievich, Tambov State Technical University Elena Nemtseva, Marina Gerasimova, Siberian Federal University, Krasnoyarsk Characterization of two.

Spectrofluorimeter Fluorolog-3-22 (Horiba Jobin Yvon, France)

,exp ii ttI

Steady-state fluorescence Time-resolved fluorescence

Correction of fluorescence intensities for inner filter effect:

,10)

2( 470360 DD

II obscorr

I corr – the true intensity of the fluorescence, I obs - experimentally measured intensity, D360, D470 – absorption at wavelengths of 360 and 470 nm, respectively

i and i – amplitudes and the lifetimes of the i-components

The lifetimes were found from the dependence of intensity on time :

Methods: Fluorescence spectroscopy

Page 7: Tatyana Avsievich, Tambov State Technical University Elena Nemtseva, Marina Gerasimova, Siberian Federal University, Krasnoyarsk Characterization of two.

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

ANS, bound with internal sitesFree ANS ANS, bound with external sites

Fig.3 - fluorescence decays for ANS in the presence of bacterial luciferase in different mediums

Page 8: Tatyana Avsievich, Tambov State Technical University Elena Nemtseva, Marina Gerasimova, Siberian Federal University, Krasnoyarsk Characterization of two.

Issf3 –fluorescence intensity of internal binding sites (3 = 12-14 ns) 8

Iss – overall intensity of the fluorescence of the probe in the steady-state excitationIssf2 – fluorescence intensity of external binding sites (2 =4-6 ns)

Results: Deconvolution of steady-state fluorescence titration curve (Iss) into lifetime components 2 and 3

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.

Nonlinear fitting of the titration curves:

Fig. 4 - Deconvolution of ANS fluorescence titration curves at 430 nm into lifetime components (markers) and their nonlinear fitting (straight lines).

Page 9: Tatyana Avsievich, Tambov State Technical University Elena Nemtseva, Marina Gerasimova, Siberian Federal University, Krasnoyarsk Characterization of two.

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Media Viscosity, cP2 -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

Table 2. – Characteristics of 1,8-ANS binding to the bacterial luciferase

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.

Results: influence of viscous media on the binding characteristics of the protein

Page 10: Tatyana Avsievich, Tambov State Technical University Elena Nemtseva, Marina Gerasimova, Siberian Federal University, Krasnoyarsk Characterization of two.

Fig. 5 – The model of two emmiting states of 1,8-ANS[Gufeng at al., Biochimica et Biophysica Acta., 2006]

460-480 nm

510-540 nm

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.

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Discussion: mechanisms of ANS interaction with luciferase

Page 11: Tatyana Avsievich, Tambov State Technical University Elena Nemtseva, Marina Gerasimova, Siberian Federal University, Krasnoyarsk Characterization of two.

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

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Fig. 4 – CPK representation of bacterial luciferase molecule (PDB id: 3FGC): hydrophobic (green), positivelycharged (indigo) and other (white) amino acid residuals,

FMN in active center is red.

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 Preferential hydration of proteins excess

water leads to the quenching of the probe the surface charge of the luciferase is changed no binding

Discussion: mechanisms of ANS interaction with luciferase

Page 12: Tatyana Avsievich, Tambov State Technical University Elena Nemtseva, Marina Gerasimova, Siberian Federal University, Krasnoyarsk Characterization of two.

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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Conclusions: