# Charge Transport Phenomena in Peptide Molecular Junctions

Authors: Alessandra Luchini; David P. Long; Iosif I. Vaisman; Emanuel F. Petricoin; David H. Geho; Lance A. Liotta
Journal: Research Letters in Nanotechnology
 (2008)

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## Abstract

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.


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## Body

## 1. Introduction

The
incorporation of biomolecules into nanoscale molecular junctions has become an
area of intense research relevant to molecular electronics [1, 2]. Nevertheless,
understanding electron transport through the complex structure of biomolecules
has been difficult, and at times contradictory [3, 4]. Inelastic
electron tunneling spectroscopy (IETS) is a powerful nanoscopic tool for
characterizing the interaction between tunneling electrons and discrete
molecular vibrations along the pathway of conduction [5]. Recently, IETS has been used to
identify incorporated molecular species [6], chemical interactions
at the metal-molecule interface [7], orientation of the
molecule [8], and the orbital
pathway followed by tunneling electrons [9]. While electron transport properties of
proteins have been a matter of interest for some time [10–13], IETS has never been applied to the
characterization of molecular junctions containing large protein polypeptides. Here, we investigate whether IETS is
applicable to polypeptide-based molecular junctions and can detect
physiologically relevant posttranslational
modifications of a peptide such as phosphorylation. In this study, we incorporate a peptide and its
phosphorylated derivative in a well-described molecular junction
architecture [7, 14] and characterize them
using IETS and I/V analysis.


## 2. Results and Discussion

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 [6]. An eleven
amino acid sequence from the regulatory domain of CaM  kinase
II (amino acids 281–291) was utilized as a test peptide. As
a basis for in
silico structural studies, the coordinates for
amino acids 281–291 were obtained from the
crystallographic structure of CaM kinase
II PDB 2bdw [15].Thr 286 was found within the helical portion of the
isolated test [15]. After
substituting Ile 281 with Met and Asp 285 with Glu, energy minimization was
performed using conjugate gradient method on the following amino acid sequence:
Met-His-Arg-Gln-Glu-Thr-Val-Asp-Cys-Leu-Lys.  In the minimized structure, the side chain of Thr 286 was phosphorylated and the modified peptide was
further minimized using the same algorithm (CHARMm force field [16]). The minimization was
carried out with no solvent and with the implicit solvent represented by
distance-dependent dielectric. The nonphosphorylated test peptide is a
combination of random coil and alpha helical secondary structural elements
(Figure 1(a)). The model for the phosphorylated
peptide (Figure 1(b)) revealed a marked conformational
alteration.  The interatom
distance between C(b) of Arg 283 and O(g) of Thr 286 in the nonphosphorylated peptide is 3.310 Å, while it is 5.214 Å in the
phosphorylated peptide, an increased distance of 1.904 Å.(a) Nonphosphorylated CaM
kinase II-derived peptide and
(b) phosphorylated peptide. Ribbon, ball and stick, and wireframe
3D structure representations.
(a)(b)The test peptide was
characterized using a self-assembled metal-peptide-metal junction [14]. The
peptide’s cysteine provided a thiol moiety for linkage to the gold electrode [17, 18]. The difference in
molecular weight between nonphosphorylated and phosphorylated peptides is 5.5%, and the difference in molecular
volume for the minimized conformations of the two forms of the peptide is in
the range of 3 to 8%, depending on the way the volume is calculated. Despite
the conformational dissimilarity, the incremental changes in volume and overall
shape of the peptide lead to very similar packing densities on the electrode.
The estimated area of contact in a microsphere junction
is 60 nm2. The “footprint" of a single CaM Kinase II peptide
molecule on an electrode is 1–3 nm2, depending on the
orientation, thus 20 to 60 molecules (either nonphosphorylated or
phosphorylated) can be attached to a 60 nm2 surface area.Magnetic arrays were cleaned as previously described [19]. Arrays were modified with CaM kinase (MHRQETVDCLK, Anaspec, San Jose, CA) and phosphorylated CaM kinase
(MHRQEpTVDCLK, Anaspec, San Jose, CA) by incubating in 2 mL of 10 μM solution in MilliQ water at 4°C for a
duration not less than 24 hours.  The
substrates were then rigorously rinsed in MilliQ water followed by drying. Using
magnetic assembly, metallized spheres were deposited at the source/drain gap,
completing the electrical circuit through the peptides. Incorrectly seated assemblies
could be detected by metal-metal contacts, which were easily excluded from the
data set (not shown).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 [19]. IETS provided a
means to measure the vibrational modes of the metal-protein-metal junction [19–22]. These studies were performed at 4 K with standard ac modulation techniques combined with two lock-in
amplifiers to record the second harmonic signal (d2I/dV2) which was then normalized by
the differential conductance (dI/dV) to yield the IET spectrum of junctions
containing either the nonphosphorylated or the phosphorylated
peptides (Figure 2). Fourier transform infrared spectra (FT-IR)
were obtained on a spectrum
RX I FT-IR spectrometer (Perkinelmer, Wellesley, MA) at a resolution of 4 cm-1 at room temperature. Reference
spectra of air were recorded and subtracted from the sample spectra.  CaM kinase
II derived peptides were dissolved in anhydrous isopropanol (100 μg/mL). The solution was dried on the
surface of KBr plates under N2.Characteristic IETS spectra (bolder line) for the nonphosphorylated
(a) and phosphorylated (b) peptides.  FTIR
results of each peptide are shown beneath the IET spectra.
(a)(b)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 [23]. IETS peak 6 encompasses a number
of modes detected via FT-IR, including the amide A and amide B bands [23]. Amino acid side chains also
contribute to the IET spectra.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 [23].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 [24]. In the IET spectra, there is a
slight red shift in frequency in peak 2 in the nonphosphorylated
peptide when compared to the phosphorylated peptide that matches the location
of the P-OC stretch detected by FT-IR. Taken together, the IETS results
for the phosphorylated and nonphosphorylated
peptides indicate that the peptide backbone and side chains contribute to
electron tunneling in the peptide.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 (Figure3(a)). The distribution of current
values at 500 mV showed a major peak (Figure 3(b)).(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).
(a)(b)(c)(d)(e)(f)Proposed mechanisms of transport
through alpha helices include: (1) the electrostatic fields
created by the dipole moment of peptide helices; (2)π orbitals of the peptide backbone present within helices; and (3) through hydrogen bonds that
contribute to the structural stability of helices [11, 13, 25–27]. Electron
transport through phosphorylated CaM kinase II-derived peptide was reproducibly
observed in a large number (62) of molecular electronics devices (Figure 3(c)). The reproducible average current
value at 0.5 V was 6 nA, an order of magnitude
less than that found with the nonphosphorylated
peptide (62 nA).The
distribution of current values at 500 mV showed a major peak (Figure3(d))
and electron transport through the immobilized phosphorylated peptide was very
reproducible (Figure 3(e)).
Helix
denaturization has previously been shown to alter the rate of electron
transport in a dichromomorphic peptide model, resulting in an order of
magnitude difference in electron transport [28]. A reproducible 10-fold difference
in electron transport between the phosphorylated and nonphosphorylated
form of CaM kinase II-derived peptide is shown in Figure 3(f). Since the calculated packing
densities of the two forms of the peptide are very similar and thus the number
of molecules analyzed in each junction is comparable, this difference is likely
attributable to a conformational shift within the secondary structure of the
peptide.


## 3. Conclusion

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.



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*Source: 853253-2008-03-26.xml*