Escape-time electrometry is a recently developed experimental technique that offers the ability to measure the effective electrical charge of a single biomolecule in solution with sub-elementary charge precision. The approach relies on measuring the average escape-time of a single charged macromolecule or molecular species transiently confined in an electrostatic fluidic trap. Comparing the experiments with the predictions of a mean-field model of molecular electrostatics, we have found that the measured effective charge even reports on molecular conformation, e.g., folded or disordered state, and non-uniform charge distribution in disordered proteins or polyelectrolytes. Here we demonstrate the ability to use the spectral dimension to distinguish minute differences in electrical charge between individual molecules or molecular species in a single simultaneous measurement, under identical experimental conditions. Using one spectral channel for referenced measurement, this kind of photophysical distinguishability essentially eliminates the need for accurate knowledge of key experimental parameters, otherwise obtained through intensive characterization of the experimental setup. As examples, we demonstrate the ability to detect small differences (∼5%) in the length of double-stranded DNA fragments as well as single amino acid exchange in an intrinsically disordered protein, prothymosin α.
REFERENCES
1.
M. F.
Perutz
, “Electrostatic effects in proteins
,” Science
201
, 1187
–1191
(1978
).2.
A.
Wada
and H.
Nakamura
, “Nature of the charge distribution in proteins
,” Nature
293
, 757
–758
(1981
).3.
D. B.
Thompson
, J. J.
Cronican
, and D. R.
Liu
, “Engineering and identifying supercharged proteins for macromolecule delivery into mammalian cells
,” Methods Enzymol.
503
, 293
–319
(2012
).4.
M. S.
Lawrence
, K. J.
Phillips
, and D. R.
Liu
, “Supercharging proteins can impart unusual resilience
,” J. Am. Chem. Soc.
129
, 10110
–10112
(2007
).5.
S.
Mirceta
, “Evolution of mammalian diving capacity traced by myoglobin net surface charge
,” Science
340
, 1234192
(2013
).6.
S. C. L.
Kamerlin
, P. K.
Sharma
, R. B.
Prasad
, and A.
Warshel
, “Why nature really chose phosphate
,” Q. Rev. Biophys.
46
, 1
–132
(2013
).7.
A.
Bah
, R. M.
Vernon
, Z.
Siddiqui
, M.
Krzeminski
, R.
Muhandiram
, C.
Zhao
, N.
Sonenberg
, L. E.
Kay
, and J. D.
Forman-Kay
, “Folding of an intrinsically disordered protein by phosphorylation as a regulatory switch
,” Nature
519
, 106
–109
(2015
).8.
A. M.
Bode
and Z. G.
Dong
, “Post-translational modification of p53 in tumorigenesis
,” Nat. Rev. Cancer
4
, 793
–805
(2004
).9.
L.
Martin
, X.
Latypova
, and F.
Terro
, “Post-translational modifications of tau protein: Implications for Alzheimer’s disease
,” Neurochem. Int.
58
, 458
–471
(2011
).10.
J. T. G.
Overbeek
and M. J.
Voorn
, “Phase separation in polyelectrolyte solutions. Theory of complex coacervation
,” J. Cell. Comp. Physiol.
49
, 7
–26
(1957
).11.
W. M.
Aumiller
and C. D.
Keating
, “Phosphorylation-mediated RNA/peptide complex coacervation as a model for intracellular liquid organelles
,” Nat. Chem.
8
, 129
–137
(2016
).12.
G. S.
Manning
, “Limiting laws and counterion condensation in polyelectrolyte solutions. I. colligative properties
,” J. Chem. Phys.
51
, 924
–933
(1969
).13.
G. S.
Manning
, “Molecular theory of polyelectrolyte solutions with applications to electrostatic properties of polynucleotides
,” Q. Rev. Biophys.
11
, 179
–246
(1978
).14.
G. S.
Manning
, “Electrostatic free energies of spheres, cylinders, and planes in counterion condensation theory with some applications
,” Macromolecules
40
, 8071
–8081
(2007
).15.
R. R.
Netz
and H.
Orland
, “Variational charge renormalization in charged systems
,” Eur. Phys. J. E
11
, 301
–311
(2003
).16.
B. W.
Ninham
and V. A.
Parsegian
, “Electrostatic potential between surfaces bearing ionizable groups in ionic equilibrium with physiologic saline solution
,” J. Theor. Biol.
31
, 405
–428
(1971
).17.
S.
Alexander
, P. M.
Chaikin
, P.
Grant
, G. J.
Morales
, P.
Pincus
, and D.
Hone
, “Charge renormalization, osmotic-pressure, and bulk modulus of colloidal crystals: Theory
,” J. Chem. Phys.
80
, 5776
–5781
(1984
).18.
M.
Aubouy
, E.
Trizac
, and L.
Bocquet
, “Effective charge versus bare charge: An analytical estimate for colloids in the infinite dilution limit
,” J. Phys. A: Math. Gen.
36
, 5835
–5840
(2003
).19.
M.
Lund
and B.
Jönsson
, “Charge regulation in biomolecular solution
,” Q. Rev. Biophys.
46
, 265
–281
(2013
).20.
R. W.
O’Brien
and L. R.
White
, “Electrophoretic mobility of a spherical colloidal particle
,” J. Chem. Soc., Faraday Trans. 2
74
, 1607
–1626
(1978
).21.
J. M.
Gao
, M.
Mammen
, and G. M.
Whitesides
, “Evaluating electrostatic contributions to binding with the use of protein charge ladders
,” Science
272
, 535
–537
(1996
).22.
I.
Gitlin
, J. D.
Carbeck
, and G. M.
Whitesides
, “Why are proteins charged? Networks of charge-charge interactions in proteins measured by charge ladders and capillary electrophoresis
,” Angew. Chem., Int. Ed.
45
, 3022
–3060
(2006
).23.
J. D.
Carbeck
, J. C.
Severs
, J.
Gao
, Q.
Wu
, R. D.
Smith
, and G. M.
Whitesides
, “Correlation between the charge of proteins in solution and in the gas phase investigated by protein charge ladders, capillary electrophoresis, and electrospray ionization mass spectrometry
,” J. Phys. Chem. B
102
, 10596
–10601
(1998
).24.
F.
Ruggeri
, F.
Zosel
, N.
Mutter
, M.
Różycka
, M.
Wojtas
, A.
Ożyhar
, B.
Schuler
, and M.
Krishnan
, “Single-molecule electrometry
,” Nat. Nanotechnol.
12
, 488
–495
(2017
).25.
M.
Krishnan
, N.
Mojarad
, P.
Kukura
, and V.
Sandoghdar
, “Geometry-induced electrostatic trapping of nanometric objects in a fluid
,” Nature
467
, 692
–695
(2010
).26.
N.
Mojarad
and M.
Krishnan
, “Measuring the size and charge of single nanoscale objects in solution using an electrostatic fluidic trap
,” Nat. Nanotechnol.
7
, 448
–452
(2012
).27.
H.
Kramers
, “Brownian motion in a field of force and the diffusion model of chemical reactions
,” Physica
7
, 284
–304
(1940
).28.
M.
Krishnan
, “Electrostatic free energy for a confined nanoscale object in a fluid
,” J. Chem. Phys.
138
, 114906
(2013
).29.
M.
Krishnan
, “A simple model for electrical charge in globular macromolecules and linear polyelectrolytes in solution
,” J. Chem. Phys.
146
, 205101
(2017
).30.
K.
Gast
, H.
Damaschun
, K.
Eckert
, K.
Schulzeforster
, H. R.
Maurer
, M.
Mullerfrohne
, D.
Zirwer
, J.
Czarnecki
, and G.
Damaschun
, “Prothymosin-alpha: A biologically-active protein with random coil conformation
,” Biochemistry
34
, 13211
–13218
(1995
).31.
S.
Müller-Späth
, A.
Soranno
, V.
Hirschfeld
, H.
Hofmann
, S.
Rüegger
, L.
Reymond
, D.
Nettels
, and B.
Schuler
, “Charge interactions can dominate the dimensions of intrinsically disordered proteins
,” Proc. Natl. Acad. Sci. U. S. A.
107
, 14609
–14614
(2010
).32.
M.
Davies
, B.
Rühle
, C.
Li
, K.
Müllen
, T.
Bein
, and C.
Bräuchle
, “Insights into nanoscale electrophoresis of single dye molecules in highly oriented mesoporous silica channels
,” J. Phys. Chem. C
118
, 24013
–24024
(2014
).33.
T.
Kiuchi
, M.
Higuchi
, A.
Takamura
, M.
Maruoka
, and N.
Watanabe
, “Multitarget super-resolution microscopy with high-density labeling by exchangeable probes
,” Nat. Methods
12
, 743
–746
(2015
).34.
35.
R.
Sjoback
, J.
Nygren
, and M.
Kubista
, “Characterization of fluorescein-oligonucleotide conjugates and measurement of local electrostatic potential
,” Biopolymers
46
, 445
–453
(1998
).36.
K.
Friederich
and P.
Woolley
, “Electrostatic potential of macromolecules measured by pKa shift of a fluorophore. 1. The 3′ terminus of 16s RNA
,” Eur. J. Biochem.
173
, 227
–231
(1988
).37.
K.
Friederich
, P.
Woolley
, and K. G.
Steinhäuser
, “Electrostatic potential of macromolecules measured by pKa shift of a fluorophore. 2. Transfer RNA
,” Eur. J. Biochem.
173
, 233
–239
(1988
).© 2018 Author(s).
2018
Author(s)
You do not currently have access to this content.
