Conductivity studies of single protein molecules

A fundamental step for future uses of biomolecules in electronics is the study of the bonding, orientation and conductance of a single molecule attached to a conductive substrate, which is the building block of electronic materials and devices based on molecular conduction. This work provides an in-depth examination of morphology and electrical properties of different molecules anchored to Au(111) and to sustainable carbon materials (graphite and graphene). Cytochrome b562 (Cyt b562), TEM beta-lactamase and the superfolded green uorescent protein engineered with phenyl azide were exposed to UV irradiation to transform the azide compound into the nitrene radical, which enabled successful molecule linking to graphene. The UV-based approach was tested on the above molecules to ascertain its robustness against the speci�city of the protein used. The e�ciency of the procedure was inspected by imaging via atomic force microscopy (AFM) and scanning tunnelling microscopy (STM). By repeated sample preparation and imaging, we established suitable protein concentrations to enable single-molecule measurements on the resulting samples (e.g., the concentration range optimal for cyt b562 on gold was 0.025-0.5 �M). We used a home-built environmental cell in combination with STM to study the conductance of di�erently engineered cyt b562 proteins on Au(111), as well as the conductance of oligothiophene on gold, under di�erent humidity and temperature conditions. We found that the conductance of cyt b562 is smaller at lower relative humidity and further decreased when also temperature is reduced. Measuring the conductance as a function of the tip-substrate distance in both tip approaching and retracting modes revealed the occurrence of hysteresis. The engineered cyt b562 with two thiols in the long axis led to less hysteresis in the conductance and larger protein height on gold (from AFM) compared to the protein with thiols in the short axis. Our results stress the importance of protein engineering to control the electrical properties of functionalized surfaces. This study meets the growing demand for achieving more e�cient molecule linking to conductive substrates, and studying environmental e�ects on the electrical response of functionalized surfaces (which is relevant, e.g., to sensing applications)

Conductivity studies of single protein molecules

A fundamental step for future uses of biomolecules in electronics is the study of the bonding, orientation and conductance of a single molecule attached to a conductive substrate, which is the building block of electronic materials and devices based on molecular conduction. This work provides an in-depth examination of morphology and electrical properties of different molecules anchored to Au(111) and to sustainable carbon materials (graphite and graphene). Cytochrome b562 (Cyt b562), TEM beta-lactamase and the superfolded green uorescent protein engineered with phenyl azide were exposed to UV irradiation to transform the azide compound into the nitrene radical, which enabled successful molecule linking to graphene. The UV-based approach was tested on the above molecules to ascertain its robustness against the speci�city of the protein used. The e�ciency of the procedure was inspected by imaging via atomic force microscopy (AFM) and scanning tunnelling microscopy (STM). By repeated sample preparation and imaging, we established suitable protein concentrations to enable single-molecule measurements on the resulting samples (e.g., the concentration range optimal for cyt b562 on gold was 0.025-0.5 �M). We used a home-built environmental cell in combination with STM to study the conductance of di�erently engineered cyt b562 proteins on Au(111), as well as the conductance of oligothiophene on gold, under di�erent humidity and temperature conditions. We found that the conductance of cyt b562 is smaller at lower relative humidity and further decreased when also temperature is reduced. Measuring the conductance as a function of the tip-substrate distance in both tip approaching and retracting modes revealed the occurrence of hysteresis. The engineered cyt b562 with two thiols in the long axis led to less hysteresis in the conductance and larger protein height on gold (from AFM) compared to the protein with thiols in the short axis. Our results stress the importance of protein engineering to control the electrical properties of functionalized surfaces. This study meets the growing demand for achieving more e�cient molecule linking to conductive substrates, and studying environmental e�ects on the electrical response of functionalized surfaces (which is relevant, e.g., to sensing applications)

Functional modulation and directed assembly of an enzyme through designed non-natural post-translation modification

Post-translational modification (PTM) modulates and supplements protein functionality. In nature this high precision event requires specific motifs and/or associated modification machinery. To overcome the inherent complexity that hinders PTM's wider use, we have utilized a non-native biocompatible Click chemistry approach to site-specifically modify TEM β-lactamase that adds new functionality. In silico modelling was used to design TEM β-lactamase variants with the non-natural amino acid p-azido-L-phenylalanine (azF) placed at functionally strategic positions permitting residue-specific modification with alkyne adducts by exploiting strain-promoted azide–alkyne cycloaddition. Three designs were implemented so that the modification would: (i) inhibit TEM activity (Y105azF); (ii) restore activity compromised by the initial mutation (P174azF); (iii) facilitate assembly on pristine graphene (W165azF). A dibenzylcyclooctyne (DBCO) with amine functionality was enough to modulate enzymatic activity. Modification of TEMW165azF with a DBCO–pyrene adduct had little effect on activity despite the modification site being close to a key catalytic residue but allowed directed assembly of the enzyme on graphene, potentially facilitating the construction of protein-gated carbon transistor system