Optical and electronic coupling of the redox copper Azurin on ITO-coated quartz substrate.

We have investigated the hybrid system constituted by the redox copper protein Azurin integrated with the semiconductor indium tin oxide (ITO) coated on quartz substrate. The system appears to be a good candidate for bio-sensing and bio-optoelectronics applications, especially due to the coupling between the optical and electron transfer features of Azurin with the conductive properties and optical transparency of ITO. The optical, morphological and electrical properties of the system have been investigated by combining optical absorption and transmission, steady-state fluorescence, resonance Raman spectroscopy and scanning probe microscopies. We found that Azurin molecules are firmly anchored on ITO and retain their structural and optical features underlying the physiological electron transfer activity. Scanning tunnelling spectroscopy evidenced a good electric coupling between the protein molecules and the substrate and a concomitant modulation of the ITO semiconductor properties upon deposition of Azurin. Some interplay between the conduction and valence bands of ITO and the electronic levels of Azurin is therefore suggested. These results are of a significant relevance in the perspective of developing bio-nanodevices able to process both optical and electrical signals, in conjugation also with the biorecognition capability of the protein molecules.

Functional metalloproteins integrated with conductive substrates: detecting single molecules and sensing individual recognition events.

In the past decade, there has been significant interest in the integration of biomaterials with electronic elements: combining biological functions of biomolecules with nanotechnology offers new perspectives for implementation of ultrasensitive hybrid nanodevices. In particular, great attention has been devoted to redox metalloproteins, since they possess unique characteristics, such as electron-transfer capability, possibility of gating redox activity, and nanometric size, which make them appealing for bioelectronics applications at the nanoscale. The reliable connection of redox proteins to electrodes, aimed at ensuring good electrical contact with the conducting substrate besides preserving protein functionality, is a fundamental step for designing a hybrid nanodevice and calls for a full characterization of the immobilized proteins, possibly at the single-molecule level. Here, we describe how a multitechnique approach, based on several scanning probe microscopy techniques, may provide a comprehensive characterization of different metalloproteins on metal electrodes, disclosing unique information not only about morphological properties of the adsorbed molecules but also about the effectiveness of electrical coupling with the conductive substrate, or even concerning the preserved biorecognition capability upon adsorption. We also show how the success of an immobilization strategy, which is of primary importance for optimal integration of metalloproteins with a metal electrode, can be promptly assessed by means of the proposed approach. Besides the characterization aspect, the complementary employment of the proposed techniques deserves major potentialities for ultrasensitive detection of adsorbed biomolecules. In particular, it is shown how sensing of single metalloproteins may be optimized by monitoring the most appropriate observable. Additionally, we suggest how the combination of several experimental techniques might offer increased versatility, real-time response, and wide applicability as a detection method, once a reproducible correlation among signals coming from different single-molecule techniques is established.

Scanning tunneling spectroscopy investigation of self-assembled plastocyanin mutants onto gold substrates under controlled environment.

The study of the electronic conduction through plastocyanin (PC) mutants assembled on a gold surface has been addressed by scanning tunneling spectroscopy. The two mutants exploit a single thiol group (PCSH) or a disulfide bridge (PCSS) to covalently bind at gold surface. The I-V measurements were performed by positioning the STM tip on top of a single molecule and sweeping the bias potential between +/-1 V, under both ambient and controlled atmosphere. For PCSS, under ambient conditions, asymmetric I-V characteristics were obtained, which disappear under nitrogen atmosphere. PCSH, instead shows a symmetric I-V relation in air and under nitrogen environment. Here, as factors underlying this distinct electron conductive behaviour, a potential role for hydration water molecules and for copper redox levels are discussed.

A poplar plastocyanin mutant suitable for adsorption onto gold surface via disulfide bridge.

Aiming to achieve stable immobilization for a redox-active cupredoxin protein onto a gold substrate and its consequent molecular level monitoring by Scanning Tunnelling Microscopy (STM), we introduced a disulphide bridge within poplar plastocyanin, while avoiding the perturbation of its active site. We selected and modified residues Ile-21 to Cys and Glu-25 to Cys by structurally conservative mutagenesis. Optical absorption spectroscopy (UV-Vis), electron paramagnetic resonance (EPR), and resonance raman scattering (RRS) results indicate that the active site of the Ile21Cys, Glu25Cys plastocyanin (PCSS) to a large extent retains the spectroscopic properties of the wild-type protein. Furthermore, the redox midpoint potential of the couple CuII/CuI in PCSS, determined by cyclic voltammetry was found to be +348 mV close to the wild-type value. The STM images display self-assembled PCSS molecules immobilised onto gold substrate. Moreover, the full potentiostatic control of the electron transfer reaction during STM imaging, suggests that the adsorbed molecule maintains essentially its native redox properties.

The 1.6 A resolution crystal structure of a mutant plastocyanin bearing a 21-25 engineered disulfide bridge.

Plastocyanin is an electron-transfer protein which has been largely used for biophysical studies as well as for protein-engineering experiments. A surface disulfide bridge has been engineered in poplar plastocyanin to allow protein chemisorption on gold substrates. The mutated plastocyanin crystal structure has been studied at 1.6 A resolution (R factor = 0.145, R(free) = 0.205) to characterize the effects of the engineered disulfide on the overall protein structure and on the Cu-coordination sphere in view of biophysical applications. The new orthorhombic crystal form isolated for the mutated plastocyanin displays two protein molecules per asymmetric unit.

Morphologic maturation of tachykinin peptide-expressing cells in the postnatal rabbit retina.

Tachykinin (TK) peptides, which include substance P, neurokinin A, two neurokinin A-related peptides and neurokinin B, are widely present in the nervous system, including the retina, where they act as neurotransmitters/modulators as well as growth factors. In the present study, we investigated the maturation of TK-immunoreactive (IR) cells in the rabbit retina with the aim of further contributing to the knowledge of the development of transmitter-identified retinal cell populations. In the adult retina, the pattern of TK immunostaining is consistent with the presence of TK peptides in amacrine, displaced amacrine, interplexiform and ganglion cells. In the newborn retina, intensely immunostained TK-IR somata are located in the ganglion cell layer (GCL) and in the inner nuclear layer (INL) adjacent to the inner plexiform layer (IPL). They are characterized by an oval-shaped cell body originating a single process without ramifications. TK-IR processes are occasionally observed in the IPL and in the outer plexiform layer (OPL). Long TK-IR fiber bundles are observed in the ganglion cell axon layer. TK-IR profiles resembling small somata are rarely observed in the INL adjacent to the OPL. At postnatal day (PND) 2, some TK-IR cells display more complex morphologic features, including processes with secondary ramifications. Long TK-IR processes in the IPL are often seen to terminate with growth cones. Between PND 6 and PND 11 (eye opening), there is a dramatic increase in the number of immunolabeled processes with growth cones both in the IPL and in the OPL and the mature lamination of TK-IR fibers in laminae 1, 3 and 5 of the IPL is established. TK-IR cells attain mature morphological characteristics and the rare, putative TK-IR somata in the distal INL are no longer observed. After eye opening, growth cones are not present and the pattern typical of the adult is reached. These observations indicate that the development of TK-IR cells can be divided into an early phase (from birth to PND 6) in which these cells establish their morphological characteristics, and a later phase (from PND 6 to eye opening) in which they are involved in active growth of their processes and likely in synapse formation. Since TK peptides are thought to play neurotrophic actions in the developing nervous system and they are consistently present in the retina throughout postnatal development, they may also act as growth factors during retinal maturation.