Protein-ligand binding volume determined byFPSA, densitometry, and NMRZigmantas Toleikis§, Vladimir A. Sirotkin†, Gediminas Skvarnavicius§, Joana Smirnoviene§,Christian Roumestand‡, Daumantas Matulis§, and Vytautas Petrauskas§**Presenting author. E-mail: vytautas.petrauskas@bti.vu.lt§Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania†A.M. Butlerov Institute of Chemistry, Kazan Federal University, Kazan, Russia‡Centre de Biochimie Structurale, Universités de Montpellier, Montpellier, France

11th European Biophysical Societies’ Association (EBSA) Congress | July 16 – 20, 2017 | Edinburgh, Scotland

INTRODUCTIONWe report the values of recombinant human heat shock pro-tein 90 (Hsp90) binding volumes (i.e., the changes in proteinvolume associated with ligand binding), which were obtainedby three independent experimental techniques – fluorescentpressure shift assay (FPSA), vibrating tube densitometry, andhigh-pressure NMR. Within the error range all techniques pro-vide similar volumetric parameters of investigated protein-ligand systems.

LIGANDS

NH2 N SNH

N

N

N

Cl

N

OH

Cl

OOH

OH OH

O

O

OH

ICPD91 ICPD9 ICPD1

Cl

O

OO

OH

OH

O

OO

OH

OH

Cl

O

OO

OH

AZ1 AZ2 AZ3

OH

Cl

OH

N N

S

O

OH

OH

N N

S

O

OH

Cl

OH

O

OO

O

ICPD47 ICPD62 Radicicol

FLUORESCENT PRESSURE SHIFT ASSAY (FPSA)System of equations describing a protein dosing curve – the relationship between concentrationof added ligand, Lt, total protein concentration, Pt, and melting pressure, pm:

Lt = (exp(−∆GU/RT )− 1)

(Pt

2 exp(−∆GU/RT )+

1

exp(−∆Gb/RT )

), (1)

∆Gx = ∆G0_x + ∆Vx(pm − p0) +∆βx

2(pm − p0)

2; x = U, b, (2)

where ∆G0, ∆V and ∆β are standard state Gibbs energy, volume and compressibility factor,respectively, and indexes U and b stand for the changes related to protein unfolding and protein-ligand binding.

Flu

ore

scen

ce y

ield

(a.u

.)

1000

2000

3000

4000

p (MPa)0 100 200 300

Lt (μM of AZ2) 0 10 30 50

(a)

Δp m

(MP

a)

0

50

100

150

200

Lt (M)

0 10−6 10−5 10−4

Hsp90αN + ICPD1 AZ2 ICPD91 ICPD9

pur

e H

sp90

αN

(b)

(a)

FIGURE: (a) Unfolding profiles and (b) dosing curves of Hsp90αN.

HIGH-PRESSURE NMRThe Kd’s of Hsp90αN interaction with a ligand is calculatedfrom the chemical shift change, ∆δ, dependency on Lt and Pt:

∆δ = ∆δmaxY −

√Y 2 − 4LtPt

2Pt,

Y = Lt + Pt +Kd.

FIGURE: (a) ∆δ of Hsp90αN amino acids which were inducedby AZ3 and ICPD9 ligands. (b) ∆δ of Val92 as a function ofICPD9 concentration at 30 MPa, 120 MPa, and 150 MPa pres-sures. (c) AZ3-induced shifts in Hsp90αN (PDB ID: 1UYL).

DENSITOMETRY

The partial molar volume of a protein, V 0, and the change in protein volume associated with theligand binding, ∆Vb, are calculated using equations:

V 0 =M

d0− d− d0

Cd0, (3) V 0(R) = V 0(0) + α∆Vb, (4)

where M and C are molecular mass and molar concentration of a protein, d0 and d are densitiesof solvent and protein solution, respectively, and α is the fraction of ligand-bound protein:

α = 0.5 (1 +R+Kd/Pt)−√

0.25 (1 +R+Kd/)2 −R. (5)

Here Kd is the dissociation constant of the protein-ligand complex and R = Lt/Pt.

Solu

tio

n d

ensi

ty (g

/cm

3 )

0.9990

0.9992

0.9994

0.9996

0.9998

Ratio Lt / Pt

0 1 2 3 4

Hsp90αN + radicicol ICPD47 ICPD62 AZ3

(a)

V 0

(cm

3 /m

ol)

21360

21400

21440

21480

Ratio Lt / Pt

0 1 2 3 4

Hsp90αN + AZ3 ICPD47 ICPD62 radicicol

(b)

FIGURE: (a) Raw densitometry data and (b) the partial molar volumes of Hsp90αN at variousR.

BINDING VOLUMES

∆Vb (FPSA) ∆Vb (Densitometry) ∆Vb (NMR) Kd

ICPD91 −1± 5 cm3/mol n.d. n.d. 3× 10−5 MICPD9 −2± 5 cm3/mol n.d. 20± 4 cm3/mol 2× 10−5 MAZ3 −7± 6 cm3/mol −10± 3 cm3/mol −9± 4 cm3/mol 9× 10−5 MAZ2 −9± 6 cm3/mol n.d. n.d. 1× 10−3 MICPD1 −9± 6 cm3/mol n.d. n.d. 3× 10−5 MAZ1 −21± 11 cm3/mol n.d. n.d. 2× 10−3 MICPD47 −40± 14 cm3/mol −49± 5 cm3/mol n.d. 5× 10−9 MICPD62 n.d. −50± 4 cm3/mol n.d. 2× 10−9 Mradicicol −170± 60 cm3/mol −124± 7 cm3/mol n.d. 2× 10−10 M

ACKNOWLEDGEMENTThis research was funded by the grants S-KEL-17-56 and MIP-004/2014 from the Research Council of Lithuania.