Inelastic electron tunneling spectroscopy (IETS) is a valuable in situ spectroscopic analysis technique that provides a direct portrait of the electron transport properties of a molecular species. In the past, IETS has been applied to small molecules. Using self-assembled nanoelectronic junctions, IETS was performed for the first time on a large polypeptide protein peptide in the phosphorylated and native form, yielding interpretable spectra. A reproducible 10-fold shift of the I/V characteristics of the peptide was observed upon phosphorylation. Phosphorylation can be utilized as a site-specific modification to alter peptide structure and thereby influence electron transport in peptide molecular junctions. It is envisioned that kinases and phosphatases may be used to create tunable systems for molecular electronics applications, such as biosensors and memory devices.
The
incorporation of biomolecules into nanoscale molecular junctions has become an
area of intense research relevant to molecular electronics [
A
peptide was derived from Ca2+/calmodulin (CaM)-dependent protein kinase
II (CaM kinase II), an important mediator of
Ca2+ signaling pathways in cells.
Phosphorylation of Thr 286 induces a conformational shift that frees
this protein from Ca2+-mediated activation [
Thr 286 was found within the helical portion of the
isolated test [
(a) Nonphosphorylated CaM kinase II-derived peptide and (b) phosphorylated peptide. Ribbon, ball and stick, and wireframe 3D structure representations.
The test peptide was
characterized using a self-assembled metal-peptide-metal junction [
Magnetic arrays were cleaned as previously described [
The assembly was transferred to a
cryogenic vacuum probe station using a parametric analyzer (Agilent 4155B, Palo Alto, CA)
under computer control for I/V and IETS as
previously described [
Characteristic IETS spectra (bolder line) for the nonphosphorylated (a) and phosphorylated (b) peptides. FTIR results of each peptide are shown beneath the IET spectra.
The
amide I, II, and III bands, components of a peptide’s backbone structure, are
present within both the IETS and FT-IR spectra, as shown in IETS peak 3 [
Within
IETS peak 3 are expected modes for a number of amino acid side chains, including
Asp, Glu, Gln, Arg, Lys, and His, the peaks of which overlap with each other as
well as elements of the peptide backbone. IETS peak 4 accounts for the S-H
stretching mode of cysteine, which absorbs in a spectral region free from
overlapping by other groups [
The marked vibrational
intensity of the IETS S-H vibrational mode, compared to the lack of a prominent
FT-IR peak, is attributable to this functional group being the point of linkage
for the peptide on the metal (Au) electrode. All electrons injected into the
peptide must exit via the gold-sulfur linkage on the electrode surface making
this single bond prominent in IETS. For the phosphopeptide, a peak at
1039 cm-1 consistent with the P-OC stretch within a phosphate group
is detected by FT-IR [
I-V
traces of the nonphosphorylated CaM kinase
II-derived peptide were acquired on 74 devices, yielding an average of 62 nA at
0.5 V bias (Figure
(a) I/V properties of the molecular junctions containing the nonphosphorylated test peptide (log scale, mean and standard errors); (b) density plot of I values (A) at 0.5 V for the nonphosphorylated peptide, logarithmic scale; (c) I-V characteristics of the molecular junctions containing the phosphorylated test peptide, logarithmic scale, mean and standard errors; (d) density plot of I values (A) at 0.5 V, logarithmic scale; (e) five separate I-V traces of a molecular junction containing the phosphopeptide; (f) a comparison of the average I-V traces for plain (gray) and phosphorylated (black) test peptides (Wilcoxon rank sum p < 0.0001017).
Proposed mechanisms of transport
through alpha helices include: (1) the electrostatic fields
created by the dipole moment of peptide helices; (2)
The
distribution of current values at 500 mV showed a major peak (Figure
This work can be highly relevant to the field of molecular electronics. These data indicate that seemingly minor posttranslational modifications of a protein polypeptide can have profound effects on the electron transport properties revealed by IET spectra and I/V characteristics. Long term, the field of protein electronics may yield new classes of biomedical sensors, computing devices, and high-throughput screening tools for kinase-targeted pharmaceuticals.
The authors appreciate the generous support of Dr. Vikas Chandhoke, Mr. Tom Huff, Dr. Ranganathan Shashidhar, Dr. Enrico Garaci, Dr. Alfonso Colombatti, Dr. Claudio Belluco, Dr. Barney Bishop, Ms. Virginia Espina, and Dr. Victor Morozov. This work was partly supported by the Italian Istituto Superiore di Sanità in the framework of Italy/USA HHS cooperation agreement, George Mason University, and the Italian Ministry of Public Health.