Structure and activity of protein-nanoparticle conjugates: towards a strategy for optimizing the interface

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2008.Cataloged from PDF version of thesis.Includes bibliographical references (p. 130-145).Nanoparticle-protein conjugates have a variety of applications in imaging, sensing, assembly and control. The nanoparticle-protein interface is made of numerous complex interactions between protein side-chains and the nanoparticle surface, which are likely to affect protein structure and compromise activity. Ribonuclease S and cytochrome c are covalently linked to nanoparticles via attachment to a specific surface cysteine, with the goal of optimizing protein structure and activity, and understanding conditions that minimize non-specific adsorption. Protein behavior is explored as a function of the nanoparticle surface chemistry and material, the density of proteins on the nanoparticle surface, and the position of the labeled site. Ribonuclease S is attached to Au nanoparticles by utilizing its two-piece structure. Enzymatic activity is determined using RNA substrate with a FRET pair. Conjugation lowers the ribonucleatic activity, which is rationalized by the presence of negative charges and steric hindrance which impede RNA in reaching the active site. Cytochrome c is linked to Au and CoFe204 nanoparticles. The protein is denatured when the nanoparticle ligands are charged, but remains folded when neutral. The presence of salt in the buffer improves folding. This indicates that electrostatic interactions of charged amino acids with the charged ligands are prone to lead to protein denaturation. The attachment site can be controlled by mutations of surface residues to cysteines. Protein unfolding is more severe for nanoparticle attached in the vicinity of charged amino acids. Molecular dynamics simulations of the conjugate reveal that electrostatic interactions with· the nanoparticle ligand lead to local unfolding of [alpha]-helices of cyt c. Furthermore, the nanoparticle induces more structural disturbance when it is attached on the N- and C-terminal [alpha]-helices foldon, which is the most stable motif of cyt c and the most essential for folding.by Marie-Eve Aubin-Tam.Ph.D

Site-directed nanoparticle labeling of cytochrome c

Although nanoparticle-protein conjugates have been synthesized for numerous applications, bioconjugation remains a challenge, often resulting in denaturation or loss of protein function. This is partly because the protein–nanoparticle interface is poorly understood, which impedes the use of nanoparticles in nanomedicine. Although the effects of nanoparticle ligand and material on protein structure have been explored, the choice of the labeling site on the protein has not yet been systematically studied. To address this issue, we label cytochrome c site-specifically with a negatively charged Au nanoparticle via a covalent thiol–Au bond. The attachment site is controlled by cysteine mutations of surface residues. The effect of labeling on protein structure is probed by circular dichroism. Protein unfolding is the most severe when the nanoparticle is attached to the N- and C-terminal foldon, the core motif of cytochrome c. Also, when the nanoparticle is attached in the vicinity of charged residues, the amount of structural damage is greater because of salt-dependent electrostatic interactions with charged ligand bis(p-sulfonatophenyl) phenylphosphine on the nanoparticle. Molecular dynamics simulations also elucidate local to global structural perturbation depending on labeling site. These results suggest that the labeling site must be considered as one of the main design criteria for nanoparticle–protein conjugates

Spiral Honeycomb Microstructured Bacterial Cellulose for Increased Strength and Toughness.

Natural materials, such as nacre and silk, exhibit both high strength and toughness due to their hierarchical structures highly organized at the nano-, micro-, and macroscales. Bacterial cellulose (BC) presents a hierarchical fibril structure at the nanoscale. At the microscale, however, BC nanofibers are distributed randomly. Here, BC self-assembles into a highly organized spiral honeycomb microstructure giving rise to a high tensile strength (315 MPa) and a high toughness value (17.8 MJ m-3), with pull-out and de-spiral morphologies observed during failure. Both experiments and finite-element simulations indicate improved mechanical properties resulting from the honeycomb structure. The mild fabrication process consists of an in situ fermentation step utilizing poly(vinyl alcohol), followed by a post-treatment including freezing-thawing and boiling. This simple self-assembly production process is highly scalable, does not require any toxic chemicals, and enables the fabrication of light, strong, and tough hierarchical composite materials with tunable shape and size

The cytoplasmic domain of the AAA+ protease FtsH is tilted with respect to the membrane to facilitate substrate entry

AAA+ proteases are degradation machines that use ATP hydrolysis to unfold protein substrates and translocate them through a central pore toward a degradation chamber. FtsH, a bacterial membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. How substrates reach the FtsH central pore is an open key question that is not resolved by the available atomic structures of cytoplasmic and periplasmic domains. In this work, we used both negative stain TEM and cryo-EM to determine 3D maps of the full-length Aquifex aeolicus FtsH protease. Unexpectedly, we observed that detergent solubilization induces the formation of fully active FtsH dodecamers, which consist of two FtsH hexamers in a single detergent micelle. The striking tilted conformation of the cytosolic domain in the FtsH dodecamer visualized by negative stain TEM suggests a lateral substrate entrance between the membrane and cytosolic domain. Such a substrate path was then resolved in the cryo-EM structure of the FtsH hexamer. By mapping the available structural information and structure predictions for the transmembrane helices to the amino acid sequence we identified a linker of ∼20 residues between the second transmembrane helix and the cytosolic domain. This unique polypeptide appears to be highly flexible and turned out to be essential for proper functioning of FtsH as its deletion fully eliminated the proteolytic activity of FtsH

Crumbling Reefs and Cold-Water Coral Habitat Loss in a Future Ocean: Evidence of “Coralporosis” as an Indicator of Habitat Integrity

Ocean acidification is a threat to the net growth of tropical and deep-sea coral reefs, due to gradual changes in the balance between reef growth and loss processes. Here we go beyond identification of coral dissolution induced by ocean acidification and identify a mechanism that will lead to a loss of habitat in cold-water coral reef habitats on an ecosystem-scale. To quantify this, we present in situ and year-long laboratory evidence detailing the type of habitat shift that can be expected (in situ evidence), the mechanisms underlying this (in situ and laboratory evidence), and the timescale within which the process begins (laboratory evidence). Through application of engineering principals, we detail how increased porosity in structurally critical sections of coral framework will lead to crumbling of load-bearing material, and a potential collapse and loss of complexity of the larger habitat. Importantly, in situ evidence highlights that cold-water corals can survive beneath the aragonite saturation horizon, but in a fundamentally different way to what is currently considered a biogenic cold-water coral reef, with a loss of the majority of reef habitat. The shift from a habitat with high 3-dimensional complexity provided by both live and dead coral framework, to a habitat restricted primarily to live coral colonies with lower 3-dimensional complexity represents the main threat to cold-water coral reefs of the future and the biodiversity they support. Ocean acidification can cause ecosystem-scale habitat loss for the majority of cold-water coral reefs.BN/Marie-Eve Aubin-Tam La

Data underlying the publication: Hydrodynamic shear dissipation and transmission in lipid bilayers

This dataset includes three m-files associated with the plots for the experimental data (Figure 2 in the manuscript) and numerical simulations (Figure 4 in the manuscript). The associated mat and txt files containing the data are included. The m-files will call these files