Transferrin, a central player in iron transport, has been recognized not only for its role in binding iron but also for its interaction with other metals, including titanium. This study employs solid-state nanopores to investigate the binding of titanium ions [Ti(IV)] to transferrin in a single-molecule and label-free manner. We demonstrate the novel application of solid-state nanopores for single-molecule discrimination between apo-transferrin (metal-free) and Ti(IV)-transferrin. Despite their similar sizes, Ti(IV)-transferrin exhibits a reduced current drop, attributed to differences in translocation times and filter characteristics. Single-molecule analysis reveals Ti(IV)-transferrin’s enhanced stability and faster translocations due to its distinct conformational flexibility compared to apo-transferrin. Furthermore, our study showcases solid-state nanopores as real-time monitors of biochemical reactions, tracking the gradual conversion of apo-transferrin to Ti(IV)-transferrin upon the addition of titanium citrate. This work offers insights into Ti(IV) binding to transferrin, promising applications for single-molecule analysis and expanding our comprehension of metal–protein interactions at the molecular level.

1.
N.
Abbaspour
,
R.
Hurrell
, and
R.
Kelishadi
, “
Review on iron and its importance for human health
,”
J. Res. Med. Sci.
19
(
2
),
164
(
2014
); available at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3999603/
2.
R. J.
Wood
et al,
Modern Nutrition in Health and Disease, Lippincott’s Illustrated Reviews Biochemistry
(
Lippincott Williams & Wilkins
,
2005
), pp.
248
270
.
3.
L. R.
McDowell
,
Minerals in Animal and Human Nutrition
(
Academic Press, Inc.
,
1992
).
4.
R.
Yip
and
P. R.
Dallman
, in
Iron: Present Knowledge in Nutrition
, edited by
B. A.
Bowman
and
R. M.
Russell
(
Springer
,
1996
), pp.
311
328
.
5.
H. A.
Huebers
and
C. A.
Finch
, “
The physiology of transferrin and transferrin receptors
,”
Physiol. Rev.
67
(
2
),
520
582
(
1987
).
6.
P.
Aisen
and
E. B.
Brown
, “
Structure and function of transferrin
,”
Prog. Hematol.
9
,
25
56
(
1975
); available at https://pubmed.ncbi.nlm.nih.gov/766075/
7.
H. A.
Huebers
and
C. A.
Finch
, “
Transferrin: Physiologic behavior and clinical implications
,”
Blood
64
,
763
767
(
1984
).
8.
C.-B.
Laurell
and
B.
Ingelman
, “
The iron-binding protein of swine serum
,”
Nutr. Rev.
43
(
7
),
209
211
(
1985
).
9.
A. D.
Tinoco
and
A. M.
Valentine
, “
Ti(IV) binds to human serum transferrin more tightly than does Fe(III)
,”
J. Am. Chem. Soc.
127
(
32
),
11218
11219
(
2005
).
10.
R. B.
Martin
et al, “
Transferrin binding of Al3+ and Fe3+
,”
Clin. Chem.
33
,
405
(
1987
); available at https://pubmed.ncbi.nlm.nih.gov/3815806/
11.
H.
Brock
, “
Transferrins
,” in
Metalloproteins, Part 2
, edited by
P. M.
Harrison
(
Verlag Chemie
,
Weinheim
,
1995
), pp.
183
261
.
12.
H.
Sun
et al, “
The first specific TiIV–protein complex: Potential relevance to anticancer activity of titanocenes
,”
Angew. Chem., Int. Ed.
37
(
11
),
1577
1579
(
1998
).
13.
J. C.
Cannon
and
N. D.
Chasteen
, “
Nonequivalence of the metal binding sites in vanadyl-labeled human serum transferrin
,”
Biochemistry
14
(
21
),
4573
4577
(
1975
).
14.
P.
Aisen
,
R.
Aasa
, and
A. G.
Redfield
, “
The chromium, manganese, and cobalt complexes of transferrin
,”
J. Biol. Chem.
244
(
17
),
4628
4633
(
1969
).
15.
J. B.
Vincent
and
S.
Love
, “
The binding and transport of alternative metals by transferrin
,”
Biochim. Biophys. Acta, Gen. Subj.
1820
(
3
),
362
378
(
2012
).
16.
J. P.
Curtin
et al, “
The role of citrate, lactate and transferrin in determining titanium release from surgical devices into human serum
,”
JBIC, J. Biol. Inorg. Chem.
23
,
471
480
(
2018
).
17.
L.
Ciavatta
et al, “
On the hydrolysis of the titanium(IV) ion in chloride media
,”
Polyhedron
4
(
1
),
15
22
(
1985
).
18.
A. E.
Martell
and
R. M.
Smith
,
Critical Stability Constants
(
Plenum Press
,
New York
,
1974
), Vol.
1
.
19.
L.
Xue
et al, “
Solid-state nanopore sensors
,”
Nat. Rev. Mater.
5
(
12
),
931
951
(
2020
).
20.
Y.
Luo
et al, “
Application of solid-state nanopore in protein detection
,”
Int. J. Mol. Sci.
21
(
8
),
2808
(
2020
).
21.
M.
O’Donohue
et al, “
Use of a solid-state nanopore for profiling the transferrin receptor protein and distinguishing between transferrin receptor and its ligand protein
,”
Electrophoresis
44
(
1–2
),
349
359
(
2023
).
22.
J.
Sha
et al, “
Identification of spherical and nonspherical proteins by a solid-state nanopore
,”
Anal. Chem.
90
(
23
),
13826
13831
(
2018
).
23.
F.
Piguet
et al, “
Identification of single amino acid differences in uniformly charged homopolymeric peptides with aerolysin nanopore
,”
Nat. Commun.
9
(
1
),
966
(
2018
).
24.
P.
Martin-Baniandres
et al, “
Enzyme-less nanopore detection of post-translational modifications within long polypeptides
,”
Nat. Nanotechnol.
18
,
1335
(
2023
).
25.
J.
Saharia
et al, “
Molecular-level profiling of human serum transferrin protein through assessment of nanopore-based electrical and chemical responsiveness
,”
ACS Nano
13
(
4
),
4246
4254
(
2019
).
26.
J.
Saharia
et al, “
Modulation of electrophoresis, electroosmosis and diffusion for electrical transport of proteins through a solid-state nanopore
,”
RSC Adv.
11
(
39
),
24398
24409
(
2021
).
27.
J.
Saharia
,
Y. M.
Nuwan
,
D. Y.
Bandara
, and
M. J.
Kim
, “
Investigating protein translocation in the presence of an electrolyte concentration gradient across a solid-state nanopore
,”
Electrophoresis
43
(
5–6
),
785
792
(
2022
).
28.
K. J.
Freedman
et al, “
Solid-state nanopore detection of protein complexes: Applications in healthcare and protein kinetics
,”
Small
9
(
5
),
750
759
(
2013
).
29.
K. J.
Freedman
et al, “
Single molecule unfolding and stretching of protein domains inside a solid-state nanopore by electric field
,”
Sci. Rep.
3
(
1
),
1638
(
2013
).
30.
K. J.
Freedman
et al, “
Nonequilibrium capture rates induce protein accumulation and enhanced adsorption to solid-state nanopores
,”
ACS Nano
8
(
12
),
12238
12249
(
2014
).
31.
K. J.
Freedman
et al, “
Chemical, thermal, and electric field induced unfolding of single protein molecules studied using nanopores
,”
Anal. Chem.
83
(
13
),
5137
5144
(
2011
).
32.
J.
Saharia
et al, “
Assessment of 1/f noise associated with nanopores fabricated through chemically tuned controlled dielectric breakdown
,”
Electrophoresis
42
(
7–8
),
899
909
(
2021
).
33.
J.
Saharia
et al, “
Over one million DNA and protein events through ultra-stable chemically-tuned solid-state nanopores
,”
Small
19
,
2300198
(
2023
).
34.
Y. M.
Nuwan
,
D. Y.
Bandara
et al, “
Beyond nanopore sizing: Improving solid-state single-molecule sensing performance, lifetime, and analyte scope for omics by targeting surface chemistry during fabrication
,”
Nanotechnology
31
(
33
),
335707
(
2020
).
35.
H.
Kwok
,
K.
Briggs
, and
V.
Tabard-Cossa
, “
Nanopore fabrication by controlled dielectric breakdown
,”
PLoS One
9
(
3
),
e92880
(
2014
).
36.
J. M.
Collins
et al, “
Titanium(IV) citrate speciation and structure under environmentally and biologically relevant conditions
,”
Inorg. Chem.
44
(
10
),
3431
3440
(
2005
).
37.
Y. F.
Deng
et al, “
Speciation of water-soluble titanium citrate: Synthesis, structural, spectroscopic properties and biological relevance
,”
Polyhedron
26
(
8
),
1561
1569
(
2007
).
38.
Y. M.
Bandara
,
D. Y.
Nuwan
et al, “
Nanopore data analysis: Baseline construction and abrupt change-based multilevel fitting
,”
Anal. Chem.
93
(
34
),
11710
11718
(
2021
).
39.
B.
Ledden
et al,
Nanopores: Sensing and Fundamental Biological Interactions
(
Springer
,
2011
), pp.
129
150
.
40.
A. A.
Rashin
,
M.
Iofin
, and
B.
Honig
, “
Internal cavities and buried waters in globular proteins
,”
Biochemistry
25
(
12
),
3619
3625
(
1986
).
41.
R.
Phillips
et al,
Physical Biology of the Cell
(
Garland Science
,
2012
).
42.
W.
Si
and
A.
Aksimentiev
, “
Nanopore sensing of protein folding
,”
ACS Nano
11
(
7
),
7091
7100
(
2017
).
43.
W. B.
Dunbar
, “
Comment on accurate data process for nanopore analysis
,”
Anal. Chem.
87
(
20
),
10650
10652
(
2015
).
44.
C.
Wen
,
D.
Dematties
, and
S.-L.
Zhang
, “
A guide to signal processing algorithms for nanopore sensors
,”
ACS Sens.
6
(
10
),
3536
3555
(
2021
).
45.
D.
Rodriguez-Larrea
and
H.
Bayley
, “
Multistep protein unfolding during nanopore translocation
,”
Nat. Nanotechnol.
8
(
4
),
288
295
(
2013
).
46.
C.
Merstorf
et al, “
Wild type, mutant protein unfolding and phase transition detected by single-nanopore recording
,”
ACS Chem. Biol.
7
(
4
),
652
658
(
2012
).
47.
A.
Asandei
et al, “
Nanoscale investigation of generation 1 PAMAM dendrimers interaction with a protein nanopore
,”
Sci. Rep.
7
(
1
),
6167
(
2017
).
48.
E. C.
Yusko
et al, “
Single-particle characterization of Aβ oligomers in solution
,”
ACS Nano
6
(
7
),
5909
5919
(
2012
).
49.
L.
Mereuta
et al, “
Protein nanopore-based, single-molecule exploration of copper binding to an antimicrobial-derived, histidine-containing chimera peptide
,”
Langmuir
28
,
17079
17091
(
2012
).

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