Self-assembling peptide and protein amyloids: from structure to tailored function in nanotechnology

Self-assembled peptide and protein amyloid nanostructures have traditionally been considered only as pathological aggregates implicated in human neurodegenerative diseases. In more recent times, these nanostructures have found interesting applications as advanced materials in biomedicine, tissue engineering, renewable energy, environmental science, nanotechnology and material science, to name only a few fields. In all these applications, the final function depends on: (i) the specific mechanisms of protein aggregation, (ii) the hierarchical structure of the protein and peptide amyloids from the atomistic to mesoscopic length scales and (iii) the physical properties of the amyloids in the context of their surrounding environment (biological or artificial). In this review, we will discuss recent progress made in the field of functional and artificial amyloids and highlight connections between protein/peptide folding, unfolding and aggregation mechanisms, with the resulting amyloid structure and functionality. We also highlight current advances in the design and synthesis of amyloid-based biological and functional materials and identify new potential fields in which amyloid-based structures promise new breakthroughs

Dual-spectral interferometric sensor for quantitative study of protein-DNA interactions

Thesis (Ph.D.)--Boston UniversityThe maintenance and functions of the genome are facilitated by DNA-binding proteins, whose specific binding mechanisms are not yet fully understood. Recently, it was discovered that the recognition and capture ofDNA conformational flexibility and deformation by DNA-binding proteins serve as an indirect readout mechanism for specific recognition and facilitate important cellular functions. Various biophysical techniques have been employed to elucidate this conformational specificity of protein-DNA interactions. These techniques are not sufficiently high-throughput to perform systematic investigation ofvarious protein-DNA complexes and their functions. Microarray-based high-throughput methods enable large-scale and comprehensive evaluation of the binding affmities of protein-DNA interactions, but do not provide conformational information. In this dissertation, we developed a tool that enables high-throughput quantification of both conformational specificity and binding affinity of protein-DNA interactions. Our approach is to combine quantitative detection of DNA conformational change and protein-DNA binding in a DNA microarray format. The DNA conformational change is measured by spectral self-interference fluorescence microscopy that determines surface-immobilized DNA conformation by measuring axial height offluorophores tagged to specific nucleotides. The amount of bound protein and DNA are measured by white light reflectance spectroscopy that quantifies molecular surface densities by measuring bioniolecule layer thicknesses. By implementing a dual-spectral imaging configuration, we can perform the two independent interferometric measurements in parallel using two separate spectral bandwidths. [TRUNCATED

Dual-spectral interferometric sensor for quantitative study of protein-DNA interactions

Thesis (Ph.D.)--Boston UniversityThe maintenance and functions of the genome are facilitated by DNA-binding proteins, whose specific binding mechanisms are not yet fully understood. Recently, it was discovered that the recognition and capture ofDNA conformational flexibility and deformation by DNA-binding proteins serve as an indirect readout mechanism for specific recognition and facilitate important cellular functions. Various biophysical techniques have been employed to elucidate this conformational specificity of protein-DNA interactions. These techniques are not sufficiently high-throughput to perform systematic investigation ofvarious protein-DNA complexes and their functions. Microarray-based high-throughput methods enable large-scale and comprehensive evaluation of the binding affmities of protein-DNA interactions, but do not provide conformational information. In this dissertation, we developed a tool that enables high-throughput quantification of both conformational specificity and binding affinity of protein-DNA interactions. Our approach is to combine quantitative detection of DNA conformational change and protein-DNA binding in a DNA microarray format. The DNA conformational change is measured by spectral self-interference fluorescence microscopy that determines surface-immobilized DNA conformation by measuring axial height offluorophores tagged to specific nucleotides. The amount of bound protein and DNA are measured by white light reflectance spectroscopy that quantifies molecular surface densities by measuring bioniolecule layer thicknesses. By implementing a dual-spectral imaging configuration, we can perform the two independent interferometric measurements in parallel using two separate spectral bandwidths. [TRUNCATED

Optical Properties and Application of Metallic Nanoparticles and their Assembled Superstructures.

This dissertation reports the development of novel targeted contrast agents based on gold nanorods (GNRs) and their application in imaging cancer cells, cardiovascular inflammatory diseases and drug delivery monitoring using photoacoustic imaging (PAI). The GNRs based contrast agents were imaged with high signal-to-noise ratio (~17) and excellent spatial resolution (~250 micron) with concentration down to 10pM in biological tissues. The inherent disadvantage of limited imaging depth (~5 mm from skin surface) in PAI due to strong attenuation of light in biological tissues restricts the monitoring of drug delivery in intra-articular connective tissues. A novel targeted optical and nuclear dual-modality contrast agent was successfully developed by radiolabeling GNRs with [125I] to monitor anti-rheumatic drug delivery. PAI and nuclear imaging in combination present the detailed distribution of GNRs conjugated anti-rheumatic agent in intra-articular connective tissues with concentration down to 10 pM and radioactive label of 5 microCi . Radiolabeled contrast agents were further conjugated with polyethylene glycol (PEG) to increase the in vivo circulation time and prevent rapid clearance through accumulation into liver. Addition of PEG molecules on the surface of contrast agent increased the in vivo circulation time from 4 minutes to over 4 hours allowing specific targeting by the contrast agent. Apart from application as a contrast agent, gold nanorods due to their anisotropic shape also form interesting building blocks for 3D superstructures with wide range of optical properties for applications in plasmonics, metamaterials and sensors. Under controlled evaporation monodisperse GNRs were self-assembled into micron sized three dimensional supercrystals. The highly organized supercrystals of GNRs with plasmonic antennae enhancement of electrical field have made possible the first real-time detection of prions (concentration down to 10^-10 M in complex biological media such as serum using surface enhanced raman scattering. CdTe nanoparticle-GNRs superstructures were made through biotin-streptavidin specific binding and investigated as potential gain materials for overcoming metallic losses in metamaterials through exciton-plasmon coupling. An extremely simple technique was developed to transfer gold nanorods from aqueous to organic medium, which allowed orientational ordering (order parameter of S~0.9) of low aspect ratio rods in dispersion through application of high electric fields.Ph.D.Chemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/78935/1/agashish_1.pd

Digital control of protein-nucleic acid interactions with self-assembled DNA nanosystems

Understanding how enzymes access, transform or degrade a nucleic acid nanostructure is an essential step for the progress of functional DNA nanotechnology. Most approaches for the analysis of nucleic acids require enzymatic reactions. For instance sequencing-by-synthesis of confined DNA molecules is a most established approach for the analysis of the entire genomic information in living organisms. Little is known however about the physical factors that regulate enzymes functions in highly confined systems. In this doctoral thesis, I have investigated the activity of type II restriction enzymes (REs) on basic, two-dimensional DNA nanostructures (DNA origami, i.e. triangles and rectangles) that are formed upon the spontaneous hybridization of many single stranded (ss)DNA molecules over the 7,249-nt-long M13mp18 ssDNA phage genome that serves as scaffold for the inherent DNA self-assembly process. The primary action of REs is DNA fragmentation, and they are a primary defensive mechanism to viral infection in bacteria. Type II REs have an exquisite ability to specifically recognize dsDNA binding sites (termed restriction sites) and irreversibly cleave the inherent DNA molecules within the sites. They are in addition essential tools for DNA cloning in current bioengineering and biotechnology applications. On the other hand, the M13mp18 scaffold naturally possesses a number of restriction sites for each of several, known restriction enzymes. In addition, DNA origami are comprised of different structural motifs that are responsible for molecular confinement of the involved DNA molecules and the peculiar mechanical properties of the shape. My work primarily consists of an unprecedented investigation of the action of >10 type II restriction enzymes on two-dimensional (2D) DNA origami. For two enzymes in particular (HhaI and Hin1II) we fully mapped the site-specific action by activating one site at a time (by generating DNA origami mutants), and measuring the fragmentation pattern of the DNA scaffold by gel electrophoresis, after melting the DNA nanostructure. With such mutational analysis of 2D DNA origami we found that (a) restriction reactions can be efficiently inhibited in 2D DNA origami, while similar inhibition cannot be achieved with the corresponding unfolded dsDNA scaffold (as expected). We argue that the observed behaviour of dense nucleic acid architectures naturally emerges as a result of reduction in spatial degrees of freedom near restriction sites, which can be controlled through small changes to the degree of mechanical stress (e.g. torsion) near the sites. (b) In 2D DNA origami, the action of REs on a site can be predicted from the structure of the three 16 bp-long adjacent dsDNA segments, with the site located in the central one (16 bp is the distance between two consecutive crossovers that join the dsDNA segments). Specifically, a site can be cleaved only if (c) it is located 654 bp from a crossover junction, and (d) near the site, each of the adjacent dsDNA segments presents a nick in one of the two strands that form the duplex. This quantitative study reveals therefore that restriction enzyme action can be digitally controlled with the closest neighbouring 2D DNA structural pattern surrounding a restriction site. These unprecedented results also suggest how to design functional nucleic acid nanostructures, with important implications for the implementation of innovative nano-biosensor, that is briefly anticipated in the concluding section of this thesis

Qualitative and Quantitative Assessment of the 'Dangerous Activities' Categories Defined by the CISSM Controlling Dangerous Pathogens Project

The Controlling Dangerous Pathogens Project of the Center for International Security Studies at Maryland (CISSM) outlines a prototype oversight system for ongoing microbiological research to control its possible misapplication. This so-called Biological Research Security System (BRSS) foresees the creation of regional, national, and international oversight bodies that review, approve, or reject those proposed microbiological research projects that would fit three BRSS-defined categories: Potentially Dangerous Activities (PDA), Moderately Dangerous Activities (MDA), and Extremely Dangerous Activities (EDA). It is the objective of this working paper to assess these categories qualitatively and quantitatively. To do so, published US research of the years 2000-present (early- to mid-2005) will be screened for science reports that would have fallen under the proposed oversight system had it existed already. Qualitatively, these selective reports will be sorted according to the subcategories of each individual Dangerous Activity, broken down by microbiological agent, and year. Quantitatively, institutes and researchers, which conducted research that would have fallen under review by BRSS, will be listed according to category and year. Taken together, the results of this survey will give an overview of the number of research projects, institutes, and researchers that would have been affected had the new proposed system existed, and thus should allow estimating the potential impact of BRSS on US microbiological academic and industrial research in the future. Furthermore, this working paper might aid refining the proposed system

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin