TUNABLE FERRITIN BIOELECTRONICS AT THE NANOSCALE

Ferritin, the iron storage protein, which is found in human body, holds great potential for applications in molecular bioelectronics and in vivo bio-nanotechnology, since the iron core of holoferritin is semiconducting in nature. Ferritin can exhibit clear electron transfer properties, is stable within a wide range of pH and temperature, and can retain structural integrity when immobilized onto a solid surface. Inspite of appearing as a suitable candidate for broad-spectrum technological applications, some of the practical aspects, e.g., how its electronic properties can be varied/tuned, need to be better addressed. In this direction, we have successfully tested that temperature, pressure and the nature of the metal core (e.g., copper, cobalt, iron and manganese) can exert clear control on the solid-state electron transport properties of the ferritin molecules. We explored a correlation between the mechanical properties of the ferritins with their electromechanical response as the concluding part of the thesis work. For this thesis, we have adopted a multi-technique approach and have applied scanning tunneling microscopy (STM), scanning tunneling spectroscopy (STS), atomic force microscopy (AFM), atomic force spectroscopy (AFS), current-sensing atomic force spectroscopy (CSAFS), synthetic preparative methods, scanning electron microscopy (SEM), uv-visible spectrophotometry (UV-VIS), transmission electron microscopy (TEM) (with EDX) and inductively coupled plasma (ICP). The present thesis includes an introduction and four results chapters.Research was conducted under the supervision of Prof. Rupa Mukhopadhyay of the Biological Chemistry division under SBS [School of Biological Sciences]Research was carried out under DST fellowshi

Nanoscale Mechano–Electronic Behavior of a Metalloprotein as a Variable of Metal Content

In this work, we have explored an approach to finding a correlation between the mechanical response of a metalloprotein against a range of applied force (by force curve analysis) and its electrical response under pressure stimulation (by current sensing atomic force spectroscopy) at the nanoscale. Iron-storage protein ferritin has been chosen as an experimental model system because it naturally contains a semiconducting iron core. This core consists of a large number of iron atoms and is therefore expected to exert a clear influence on the overall mechanical response of the protein structure. Four different ferritins (apoferritin, Fe­(III)-ferritins containing ∼750 and ∼1400 iron atoms, and holoferritin containing ∼2600 iron atoms) were chosen in order to identify any relation between the mechano–electronic behavior of the ferritins and their metal content. We report the measurement of Young’s modulus values of the ferritin proteins as applicable in a nanoscale environment, for the first time, and show that these values are directly linked to the iron content of the individual ferritin type. The greater the iron content, the greater the Young’s modulus and in general the slower the rate of deformation against the application of force. When compressed, all the four ferritins exhibited increased electronic conductivity. A correlation between the iron content of the ferritins and the current values observed at certain bias voltages could be made at higher bias values (beyond 0.7 V), but no such discrimination among the four compressed ferritins could be made at the lower voltages. We propose that only at higher voltages can the iron atoms that reside deeper inside the core of the ferritins be accessed. The iron atoms that could be situated at the inner wall of the protein shell appear to make a general contribution to the electronic conductivity of the four ferritin systems

Rhodopsin Forms Nanodomains in Rod Outer Segment Disc Membranes of the Cold-Blooded Xenopus laevis.

Rhodopsin forms nanoscale domains (i.e., nanodomains) in rod outer segment disc membranes from mammalian species. It is unclear whether rhodopsin arranges in a similar manner in amphibian species, which are often used as a model system to investigate the function of rhodopsin and the structure of photoreceptor cells. Moreover, since samples are routinely prepared at low temperatures, it is unclear whether lipid phase separation effects in the membrane promote the observed nanodomain organization of rhodopsin from mammalian species. Rod outer segment disc membranes prepared from the cold-blooded frog Xenopus laevis were investigated by atomic force microscopy to visualize the organization of rhodopsin in the absence of lipid phase separation effects. Atomic force microscopy revealed that rhodopsin nanodomains form similarly as that observed previously in mammalian membranes. Formation of nanodomains in ROS disc membranes is independent of lipid phase separation and conserved among vertebrates

Single molecule analysis of CENP-A chromatin by high-speed atomic force microscopy

Chromatin accessibility is modulated in a variety of ways to create open and closed chromatin states, both of which are critical for eukaryotic gene regulation. At the single molecule level, how accessibility is regulated of the chromatin fiber composed of canonical or variant nucleosomes is a fundamental question in the field. Here, we developed a single-molecule tracking method where we could analyze thousands of canonical H3 and centromeric variant nucleosomes imaged by high-speed atomic force microscopy. This approach allowed us to investigate how changes in nucleosome dynamics in vitro inform us about transcriptional potential in vivo. By high-speed atomic force microscopy, we tracked chromatin dynamics in real time and determined the mean square displacement and diffusion constant for the variant centromeric CENP-A nucleosome. Furthermore, we found that an essential kinetochore protein CENP-C reduces the diffusion constant and mobility of centromeric nucleosomes along the chromatin fiber. We subsequently interrogated how CENP-C modulates CENP-A chromatin dynamics in vivo. Overexpressing CENP-C resulted in reduced centromeric transcription and impaired loading of new CENP-A molecules. From these data, we speculate that factors altering nucleosome mobility in vitro, also correspondingly alter transcription in vivo. Subsequently, we propose a model in which variant nucleosomes encode their own diffusion kinetics and mobility, and where binding partners can suppress or enhance nucleosome mobility

Probing Aberrantly Glycosylated Mucin 1 in Breast Cancer Extracellular Vesicles

Aberrantly glycosylated mucin 1 is a critical prognostic biomarker in breast epithelial cancers. Hypoglycosylated mucin 1 coats the surface of the cancer cells, where O-glycans are predominantly linked via an N-acetylgalactosamine moiety (GalNAc). Cancer cell-derived extracellular vesicles (EVs) carry biomarkers from parent cancer cells to the recipient cells and, therefore, could potentially be leveraged for diagnostics and noninvasive disease monitoring. We devised a label-free approach for identifying glycoprotein mucin 1 overexpression on breast cancer EVs. While exploring a plethora of biochemical (enzyme-linked immunosorbent assay, flow cytometry, and SDS-PAGE) and label-free biophysical techniques (circular dichroism and infrared spectroscopy (IR)) along with multivariate analysis, we discovered that mucin 1 is significantly overexpressed in breast cancer EVs and aberrant glycosylation in mucin 1 could be critically addressed using IR and multivariate analysis targeting the GalNAc sugar. This approach emerges as a convenient and comprehensive method of distinguishing cancer EVs from normal samples and holds potential for nonintrusive breast cancer liquid biopsy screening

Murine ROS disc membranes imaged at 37°C.

<p>Representative images obtained by tapping mode AFM are shown. Murine ROS disc membranes were prepared at 4°C and imaged at 37°C. Height (left) and amplitude (right) images are shown. Height images were scaled to a height range of 25 nm. Scale bar, 500 nm.</p

AFM image of an intact murine ROS disc.

<p>(A, B) Height (A) and deflection (B) images obtained by contact mode AFM generated using low force. (C, D) Height (C) and deflection (D) images obtained by contact mode AFM generated using higher force. The rim region (1) and nanodomains in the lamellar region (2) are discernible. Height images were scaled to a height range of 38 nm. Scale bar, 250 nm. Illustrations of a disc adsorbed on mica scanned by the AFM tip at low and high forces are shown next to AFM images. (E) A height profile is shown for the cross-section highlighted by a dotted line in panel C.</p

AFM images of <i>X</i>. <i>laevis</i> ROS disc membranes.

<p>(A-G) Representative deflection images of <i>X</i>. <i>laevis</i> ROS disc membranes obtained by contact mode AFM. ROS disc membranes exhibit a varying number of lobes, which are formed by deeply penetrating incisures. Scale bar, 500 nm. (H) Histogram of nanodomain sizes measured in 57 images of <i>X</i>. <i>laevis</i> ROS disc membranes. The data was fit by a Log Gaussian function (<i>n</i> = 14,390).</p

<i>X</i>. <i>laevis</i> ROS disc membrane preparation.

<p>(A) The secondary structure of rhodopsin is shown with amino acid residue differences in <i>X</i>. <i>laevis</i> (red) and murine (blue) rhodopsin highlighted. (B) Light microscopy image of purified ROS from the retina of <i>X</i>. <i>laevis</i>. Scale bar, 15 μm. (C) SDS-PAGE of <i>X</i>. <i>laevis</i> (lane 1) and murine (lane 2) ROS disc membrane preparations. The sizes of protein standards are indicated in kDa.</p