Probing hydration and mobility in enzymes by fluorescence ... · Probing hydration and mobility in enzymes by fluorescence: Implications in rational protein design Martin Hof J. Heyrovský

Probing hydration and mobility in enzymes by fluorescence: Implications in rational protein design

Martin Hof J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic

or

“Use of fluorescence spectroscopy in synthetic biology”

Probing hydration and mobility in enzymes by fluorescence: Implications in rational protein design

or

“Use of fluorescence spectroscopy in synthetic biology”

1. Introduction: synthetic biology and haloalkane dehalogenases

2. Unnatural aminoacid fluorescence in dehalogenases 3. Time-dependent fluorescence shifts in dehalogenases 4. Hydration and mobility and dehalogenases’s

enantioselectivity

Synthetic biology

One research line: Design of novel protein structures that match or improve on the functionality of existing proteins.

"Synthetic biology is a) the design and construction of new biological molecules or systems and b) the re-design of existing natural biological systems for useful purposes." Syntheticbiology.org

What is a strategy for the design of novel protein structures?

• Transplanting active sides in different protein scaffolds; reaction occurs in a “foreign” environment

• Design of the synthetic protein is mainly based on the 3D structure

But: Novel protein structures do not reach the activity of natural enzymes:

Structure alone is not enough to optimize catalytic properties

See e.g. Hilvert group Nature 503 418 (2013) or Baker group Nature 501 212 (2013)

Specifically, we are working on the Enzyme Class Haloalkane Dehalogenases (Dh)

What are the missing factors in enzyme design (beside of optimized active side 3D structure) ?

General Aim of our research to find out to what extent protein hydration and mobility close to the active site

are factors to be considered when developing new enzymes

• cleave C-X bond (X stands for Br or Cl) in more than 100 halogenated hydrocarbons & their derivatives:

• several substrates serious environmental pollutants: 1,2-dichloroethane, 1,2,3-trichloropropane, 1-chlorobutane, hexachlorocyclohexane

We are working on the Enzyme Class Haloalkane Dehalogenases

• cleave C-X bond in more than 100 halogenated hydrocarbons & their derivatives:

• enantioselectivity ability to discriminate between two enantiomers (mirror images) of a

compound with a chiral center

We are working on the Enzyme Class Haloalkane Dehalogenases

PresenterPresentation NotesEnantioselectivity was quantified by the E value, defined as the ratio of catalytic efficiency of conversion of the preferred (R) over the nonpreferred (S) enantiomer: E value = (kcat,R/Km,R)/(kcat,S/Km,S), where kcat is the catalytic constant and Km is the Michaelis constant. Racemic mixtures of 2-bromopentane, 2-bromohexane, 2-bromoheptane or ethyl 2-bromopropionate

• representatives of α/β fold hydrolase superfamily

Haloalkane Dehalogenases

Haloalkane enters the tunnel


Aspartic acid

SN2 reaction produces halogen anion


Hydrolysis produces alcohol


Dehalogenases are enantioselective


Specific Aim

?Does hydration and mobility at the access tunnel influence action (enantioselectivity) of Haloalkane Dehalogenases?

We need tools to probe site-specific hydration and mobility!

Fluorescence

Labelling strategy I: Dye is a fluorescent unnatural amino acid

incorporated into the protein matrix

Fluorescence Method I: Decomposition of steady-state spectra

Probing hydration and mobility at the access tunnel of haloalkane dehalogenases by fluorescence

Dye is a fluorescent unnatural amino acid incorporated into the protein matrix

OHO O

COOHH2N

DhaA has narrow tunnel mouth DbjA has wide tunnel mouth

L-(7-hydroxycoumarin-4-yl)ethylglycine


OHO O

COOHH2N



DhaA:C176UAA DbjA:G183UAA

How to get the UAA into the protein?


OHO O

COOHH2N

Bacteria need • L-(7-hydroxycoumarin-4-yl)ethylglycine • orthogonal tRNA and aminoacyl-tRNA synthetase pair • Modified plasmids DhaA:C176TAG and DbjA:G183TAG (Cys176 and Gly183

replaced by the TAG stop codon, respectively)

Labelling strategy I: Dye is a fluorescent unnatural amino acid

incorporated into the protein matrix

Fluorescence Method I: Decomposition of steady-state spectra

Excited state

OHO O

COOHH2N

Photophysics of the UAA: How to measure hydation? Steady-state fluorescence emission is due to different emitting species

Choudhury, S. D.; Nath, S.; Pal, H. J. Phys. Chem. B 2008, 112, 7748.

UAA spectrum is sensitive to water content and can sense structured water Verification on AOT inverse micelles

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

350 400 450 500 550 600

0.0

0.2

0.4

0.6

0.8

1.0

C

B

Rel

ativ

e in

tens

ity (a

.u.)

Em. spectrum in AOT, w0 = 0

fit - neutral formA

R

elat

ive

inte

nsity

(a.u

.) Em. spectrum in AOT, w0 = 10 fit neutral form anionic form tautomer

Rel

ativ

e in

tens

ity (a

.u.)

wavelength (nm)

Em. spectrum in AOT, w0 = 40

fit neutral form anionic form tautomer

UAA spectrum is sensitive to water content and can sense structures water Verification on AOT inverse

UAA spectrum is sensitive to water content and can sense structures water Verification on AOT inverse

Excited state

Unnatural amino acid fluorescence in DhaA and DbjA


Calculations by J. Brezovsky; results summarised in M. Amaro et al. JACS 2015

Comparison of Fluorescence Data with Simulations Quantum Mechanics/Molecular Mechanics Stochastic Boundary Molecular Dynamics


Parameter DhaA

DbjA

Overall contributions of anionic and tautomeric forms to the emission steady-state spectra

UAA less hydrated

UAA more hydrated

Number of water molecules within 5 Å 9±2 21±5 or 28±4

?Why shows DbjA:G183UAA a strong tautomeric contribution?

indicating structured water

DbjA:G183UAA has two stable conformations

Unnatural amino acid fluorescence probes hydration at the access tunnel of dehalogenases

Labelling strategy and fluorescence method I:

Labelling strategy II:

Dye is part of a substrate for the enzymatic reaction, yielding a covalent enzyme-dye complex

Fluorescence Method II:

Hydration and mobility determined by time-dependent fluorescence shifts


Dye is part of a substrate for the enzymatic reaction, yielding a covalent Dh-dye complex

functional reporter reactive linker

H

O

H

OO

NO

OCl

H2N


NNH

N

O

OO

HHO

H

H

O

H

OO

NO

OCl

His272His280 Asp106

Asp103

H2N


NNH

N

O

OO

HHO

H

O

H

OO

NO

OCl

H

His272His280 Asp106

Asp103

H2N


N

O

OO

HHO

H

O

H

OO

NO

OCl

H

Asp106Asp103

H2N

Phe272Phe280


DhaA has narrow tunnel mouth Dbja has wide tunnel mouth

H

O

H

OO

NO

OCl

H2N



H

O

H

OO

NO

OCl

H2N

Labelling strategy II:

Dye is part of a substrate for the enzymatic reaction, yielding a covalent enzyme-dye complex

Fluorescence Method II:

Hydration and mobility determined by time-dependent fluorescence shifts

Time dependent fluorescence shifts monitoring solvent relaxation

Δν spectral shift

Time-Resolved Emission Spectra (TRES)

0.05 ns 0.5 ns 5 ns

Δν (cm-1)

ν(t)

kinetics τ (ns)

time

Read out parameters

total spectral shift Δν (cm-1)

relaxation time τ (ns)

Time-dependent Stokes shift ∆ ν gives directly information about the micro-polarity

• ∆ ν is directly proportional to the polarity function F

• example: C1OH: F = 0.71; ∆ ν = 2370 cm-1

C5OH: F = 0.57; ∆ ν = 1830 cm-1

Horng et al., J Phys Chem 1995 99:17311

O ON

CF3

∆ν

= ν

(t=0

) - ν

(t=∞

) in

cm-1

F = [(εs-1)/ (ε s+2)] - [(n2-1)/ (n2+2) ]

Kinetics of the SR is related to the viscosity of the microenvironment

1/T / 10-3 K-15 6 7 8 9 10 11 12

Solv

atio

n Ti

me

/ s

10-11

10-9

10-7

10-5

10-3

10-1

101

103

R. Richert et al. Chem. Phys. Lett. 1994

N

N

Ru(byp)2(CN)2

N

O

O

H

N

H

H

Tg = 92 K

170 K

τFluor = 20 ns

τCT = 4 µs

τPhosp = 0.25 s

dyes in THF 90-170 K

} } } Probed by S1→S0

Fluoreszenz

Probed by Charge-Transfer Emission

Probed by T1→T0 Phosphoreszenz

Time dependent fluorescence shifts monitoring solvent relaxation

Δν spectral shift

Time-Resolved Emission Spectra (TRES)

0.05 ns 0.5 ns 5 ns

Δν (cm-1)

ν(t)

kinetics τ (ns)

Parameter neat solvents proteins:

segment next to chomophore

total spectral shift

solvent polarity

polarity and hydration state of protein segment

relaxation time

viscosity mobility of that segment

time

• Blue-shifted steady-state spectrum of any solvatochromic dye can be due to low polarity, but also due to high viscosity!

• Only TDFS can get both information simultaneously. • Conclusions made purely on steady-state data might not

be true!!!

General remark to the use of solvatochromic dyes

See also our recent review: Amaro et al. Biophys J 2014

Transmembrane peptides might increase Laurdan probed polarity (∆ν) , but decrease mobility (τ ): steady-state “GP value” is insensitive to that!

Recent example: Machan et al Langmuir 2014: Membrane binding of several Transmembrane peptides by TDFS and steady-state “GP value”

What is known on TDFS in proteins? •Within the last 15 years: •Several theoretical attempts of understanding those TDFS (including “biological water” used by A. Zewail (Caltech)).

Many studies from various authors on TDFS in proteins show “ps to ns kinetics” (“exploration of timescale”)

S. K. Pal, A. H. Zewail, Chem. Rev. 2004, 104, 2099-2123.

What is known on TDFS in proteins?

“The TDFS is insensitive to the motion of individual water molecules. Even for solvent exposed dye (50 ps) the TDFS shows the

collective conformational protein dynamics”**

•Within the last 15 years: •Several theoretical attempts of understanding those TDFS (including “biological water” used by A. Zewail (Caltech)).

• Accepted is (in line with our ‘20 years old’ interpretation of TDFS in membranes*):

Many studies from various authors on TDFS in proteins show “ps to ns kinetics” (“exploration of timescale”)

*Hutterer et al. Biophys. Chem 1996 61 151 **Halle, B.; Nilsson, L. J. Phys. Chem. B 2009, 113,8210

How to label the protein region of interest 100 % specifically? • On what timescale do TDFS in proteins occur at a site which are

relevant for its function?

• Can we connect such TDFS data with mobility and hydration at this site?

• Can we connect such TDFS data with the function of the protein?


DhaA has narrow tunnel

mouth

DbjA has wide tunnel

mouth

H

O

H

OO

NO

OCl

H2N

TDFS data – comparison between DhaA and DbjA

∆ν (cm -1)

τr (ns) % obs.

DhaA-H272F 950 4.1 90

DbjA-H280F 1 300 2.8 70

Conclusions:

• pure nanosecond dynamics in the tunnel mouth (on the protein surface typically ~ picoseconds)

• DbjA is more hydrated and mobile when compared to DhaA (qualitatively identical result with unnatural aminoacid method)!

• Conclusions confirmed by time-resolved anisotropy and acrylamide quenching as well as other mutants

Comparison of Fluorescence Data with Simulations Quantum Mechanics/Molecular Mechanics Stochastic Boundary Molecular Dynamics

Parameter

Enzyme Interpretation

DhaA DbjA

Density of water (5 Å around dye) [kg.m-3] Dynamic Stokes shift [cm-1]

2.3 ± 0.7 950

6.0 ± 0.3 1300

Tunnel mouth is more hydrated in DbjA

Mobility expressed by B-factor of tunnel mouth near dye [Å2]

Integral relaxation time τr [ns]

112 4.1

378 2.8

Tunnel mouth is more mobile in DbjA

Residues forming the most van der Waals contacts with the dye

Phe144 Lys175

Arg179 Gly183

Calculations by J. Brezovsky; results summarised in A. Jesenska et al. JACS 2009

Can we connect such TDFS data with mobility and hydration at this site?

Are Time-Dependent Fluorescence Shifts Dependent on the Choice of the Chromophore?

•TDFS kinetics is quantitatively different •But comparative conclusions on hydration and mobility of different mutants are valid

Are Time-Dependent Fluorescence Shifts Dependent on the Choice of the Chromophore?

M. Amaro et al. J Phys Chem 2013

How to label the protein region of interest 100 % specifically? On what timescale do TDFS in proteins occur at sites which are relevant for its function? Can we connect such TDFS data with mobility and hydration at this site?

•Can we connect such TDFS data with the function of the protein?

•Can we connect such TDFS data with the function of the protein?

Specific Aim

?Does hydration and mobility at the access tunnel influence action (Enantioselectivity) of Haloalkane Dehalogenases?

Enantioselectivity towards β-bromoalkanes: DbjA versus DhaA

DbjA

DhaA

04080

120160200

E-va

lue

DhaA

DbjA

Data by Z. Prokop E-value = (kcat,R/Km,R)/(kcat,S/Km,S)

Identified all residues essential for enantioseletivity of DbjA at active site and the access tunnel

PresenterPresentation NotesRacemic mixtures of 2-bromopentane, 2-bromohexane, 2-bromoheptane or ethyl 2-bromopropionate . E value, defined as the ratio of catalytic efficiency of conversion of the preferred (R) over the nonpreferred (S) enantiomer:

Enzyme design: Transplanting the entire active site of DbjA to the scaffold of DhaA leading to DhaA12: “attempt to transplant full enantioselectivity to DhaA”

Step-wise metamorphosis of DhaA to DbjA-like enzyme:

DhaA12

Jiří Damborský (Brno)

Comparing X-ray structure of the active site and the access tunnel: DbjA versus DhaA12

Metamorphosis of DhaA into DhaA12, a mutant mimicking DbjA, was successful in the geometrical sense

Comparing enantioselectivity towards β-bromoalkanes: DbjA versus DhaA12

→Enantioselectivity of DhaA12 with β -brominated alkanes does not reach the level of DbjA

2-bromopentane2-bromohexane2-bromoheptaneethyl 2-bromopropionate

050

100150200

E-va

lue

Substrates:

TDFS data: comparison between DbjA, DhaA, DhaA12 DbjA

DhaA12

DhaA

Fluorescence Solvent Relaxation Data and Simulations

Parameter

Enzyme

Interpretation DbjA DhaA12

Residues forming the most van der Waals contacts with the dye

Arg179 Gly183

Arg171 Gly175

Contact of the dye with protein identical

Density of water (5 Å around dye) [kg.m-3] Dynamic Stokes shift [cm-1]

6.0 ± 0.3 1300

3.3 ± 0.7 1000

Microenvironment is

more hydrated in DbjA

Mobility expressed by B-factor of tunnel mouth near dye [Å2]

Integral relaxation time τr [ns]

378 2.8

158 3.8

Microenvironment is more mobile in DbjA

Calculation by J. Brezovsky

Though geometry of active

site and access tunnel is identical, substantial

differences between DbjA and DhaA12 in dynamics

and hydration at the access tunnel

↓

Thermodynamics analysis highlighting the importance of entropic contribution to DbjA

enantioselectivity

Dynamics and hydration at the access tunnel are relevant for enzyme enantioselectivity

J. Sykora et al. Nat Chem Biol 2014

Conclusions and Summary

H

O

H

OO

NO

OCl

H2NOHO O

COOHH2N

Time-dependent fluorescence shifts

Probing hydration and mobility in the enzyme

tunnel

Decomposition of UAA fluorescence spectrum

Importance of hydration and dynamics for enzyme design: engineering of nonselective enzyme DhaA towards high selectivity by transplantation of active site and access tunnel from selective DbjA region in DhaA12 is structurally comparable to DbjA, but selectivity is not obtained and dynamics and hydration of this region differs significantly

Conclusions and Summary

Acknowledgements Dr. Tatsiana Chernavets Dr.Agnieszka Olzynska, Dr. Mariana Amaro, Dr. Jan Sykora

Group of Jiří Damborský (Brno) J. Brezovsky, Z. Prokop,R. Chaloupkova Group of K. Paruch (Brno) S. Kováčová, V. Němec Academy of Sciences of the Czech R for the PRAEMIUM ACADEMIAE

Acknowledgements Dr. Tatsiana Chernavets Dr.Agnieszka Olzynska, Dr. Mariana Amaro, Dr. Jan Sykora

http://www.jh-inst.cas.cz/~fluorescence/

A. Jesenska, et al. JACS. 131 (2009) 494-501 M. Amaro, et al. J Phys Chem B 117 (2013)7898-7906 T. Koudelakova et al. Angewandte Chemie 52 (2013) 1515 M. Amaro, et al. JACS 137 (2015) 4988-4992 J. Sykora, et al. Nature Chem Biol 10 (2014) 428-430

Back home!

Matriculation at the Universität Würzburg in 1981

Probing hydration and mobility in enzymes by fluorescence: Implications in rational protein designProbing hydration and mobility in enzymes by fluorescence: Implications in rational protein designSlide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Haloalkane DehalogenasesSlide Number 9Slide Number 10Slide Number 11Slide Number 12Slide Number 13Labelling strategy I:�Dye is a fluorescent unnatural amino acid incorporated into the protein matrix ��Fluorescence Method I:�Decomposition of steady-state spectra ��Labelling strategy I:�Dye is a fluorescent unnatural amino acid incorporated into the protein matrix ��Fluorescence Method I:�Decomposition of steady-state spectra ��Slide Number 16Slide Number 17Slide Number 18Slide Number 19Labelling strategy I:�Dye is a fluorescent unnatural amino acid incorporated into the protein matrix ��Fluorescence Method I:�Decomposition of steady-state spectra ��Slide Number 21Slide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28Slide Number 29Labelling strategy II:��Dye is part of a substrate for the enzymatic reaction, yielding a covalent enzyme-dye complex��Fluorescence Method II:�Hydration and mobility determined by time-dependent fluorescence shifts��Labelling strategy II:��Dye is part of a substrate for the enzymatic reaction, yielding a covalent enzyme-dye complex��Fluorescence Method II:�Hydration and mobility determined by time-dependent fluorescence shifts��Dye is part of a substrate for the enzymatic reaction, yielding a covalent Dh-dye complexDye is part of a substrate for the enzymatic reaction, yielding a covalent Dh-dye complexDye is part of a substrate for the enzymatic reaction, yielding a covalent Dh-dye complexDye is part of a substrate for the enzymatic reaction, yielding a covalent Dh-dye complexSlide Number 36Slide Number 37Slide Number 38Labelling strategy II:��Dye is part of a substrate for the enzymatic reaction, yielding a covalent enzyme-dye complex��Fluorescence Method II:�Hydration and mobility determined by time-dependent fluorescence shifts��Slide Number 40Time-dependent Stokes shift gives directly information about the micro-polarityKinetics of the SR is related to the viscosity of the microenvironmentSlide Number 43Slide Number 44Slide Number 45Slide Number 46Slide Number 47Slide Number 48Comparison of Fluorescence Data with Simulations�Quantum Mechanics/Molecular Mechanics Stochastic Boundary Molecular Dynamics�Slide Number 50Slide Number 51Slide Number 52Slide Number 53Slide Number 54Slide Number 55Slide Number 56Slide Number 57Slide Number 58Fluorescence Solvent Relaxation Data and Simulations Slide Number 60Conclusions and SummarySlide Number 62AcknowledgementsAcknowledgementsBack home!

Documents

Probing hydration and mobility in enzymes by fluorescence ... · Probing hydration and mobility in enzymes by fluorescence: Implications in rational protein design Martin Hof J. Heyrovský