Jeffery G. Saven

Helical Wrapping of Single-Walled Carbon Nanotubes by Water Soluble Poly(p-phenyleneethynylene)

Amphiphilic, linear conjugated poly[p-{2,5-bis(3-propoxysulfonicacidsodiumsalt)}phenylene]ethynylene (PPES) efficiently disperses single-walled carbon nanotubes (SWNTs) under ultrasonication conditions into the aqueous phase. Vis-NIR absorption spectroscopy, atomic force microscopy (AFM), and transmission electron microscopy (TEM) demonstrate that these solubilized SWNTs are highly individualized. AFM and TEM data reveal that the interaction of PPES with SWNTs gives rise to a self-assembled superstructure in which a polymer monolayer helically wraps the nanotube surface; the observed PPES pitch length (13 +/- 2 nm) confirms structural predictions made via molecular dynamics simulations. This work underscores design elements important for engineering well-defined nanotube-semiconducting polymer hybrid structures.

Computational design of a protein crystal

Protein crystals have catalytic and materials applications and are central to efforts in structural biology and therapeutic development. Designing predetermined crystal structures can be subtle given the complexity of proteins and the noncovalent interactions that govern crystallization. De novo protein design provides an approach to engineer highly complex nanoscale molecular structures, and often the positions of atoms can be programmed with sub-Å precision. Herein, a computational approach is presented for the design of proteins that self-assemble in three dimensions to yield macroscopic crystals. A three-helix coiled-coil protein is designed de novo to form a polar, layered, three-dimensional crystal having the P6 space group, which has a “honeycomb-like” structure and hexameric channels that span the crystal. The approach involves: (i) creating an ensemble of crystalline structures consistent with the targeted symmetry; (ii) characterizing this ensemble to identify “designable” structures from minima in the sequence-structure energy landscape and designing sequences for these structures; (iii) experimentally characterizing candidate proteins. A 2.1 Å resolution X-ray crystal structure of one such designed protein exhibits sub-Å agreement [backbone root mean square deviation (rmsd)] with the computational model of the crystal. This approach to crystal design has potential applications to the de novo design of nanostructured materials and to the modification of natural proteins to facilitate X-ray crystallographic analysis.

Highly Tunable Photoluminescent Properties of Amphiphilic Conjugated Block Copolymers

We report a novel class of amphiphilic conjugated block copolymers composed of poly(3-octylthiophene) and poly(ethylene oxide) (POT-b-PEO) that exhibit highly tunable photoluminescence colors spanning from blue to red. POT-b-PEO self-assembles into various well-defined core/shell-type nanostructures as a result of its amphiphilicity. The self-assembly structure can be readily controlled by altering the solvent composition or by other external stimuli. The color change was completely reversible, demonstrating that the strategy can be used to manipulate the light-emission properties of conjugated polymers in a highly controllable manner without having to synthesize entirely new sets of molecules.

Theoretical and Computational Protein Design

From exponentially large numbers of possible sequences, protein design seeks to identify the properties of those that fold to predetermined structures and have targeted structural and functional properties. The interactions that confer structure and function involve intermolecular forces and large numbers of interacting amino acids. As a result, the identification of sequences can be subtle and complex. Sophisticated methods for characterizing sequences consistent with a particular structure have been developed, assisting the design of novel proteins. Developments in such computational protein design are discussed, along with recent accomplishments, ranging from the redesign of existing proteins to the design of new functionalities and nonbiological applications.

Computational design of water-soluble analogues of the potassium channel KcsA

Although the interiors of membrane and water-soluble proteins are similar in their physicochemical properties, membrane proteins differ in having larger fractions of hydrophobic residues on their exteriors. Thus, it should be possible to water-solubilize membrane proteins by mutating their lipid-contacting side chains to more polar groups. Here, a computational approach was used to generate water-soluble variants of the potassium channel KcsA. As a probe of the correctness of the fold, the proteins contain an agitoxin2 binding site from a mammalian homologue of the channel. The resulting proteins express in high yield in Escherichia coli and share the intended functional and structural properties with KcsA, including secondary structure, tetrameric quaternary structure, and tight specific binding to both agitoxin2 and a small molecule channel blocker.

Statistical and molecular dynamics studies of buried waters in globular proteins

Buried solvent molecules are common in the core of globular proteins and contribute to structural stability. Folding necessitates the burial of polar backbone atoms in the protein core, whose hydrogen‐bonding capacities should be satisfied on average. Whereas the residues in α‐helices and β‐sheets form systematic main‐chain hydrogen bonds, the residues in turns, coils and loops often contain polar atoms that fail to form intramolecular hydrogen bonds. The statistical analysis of 842 high resolution protein structures shows that well‐resolved, internal water molecules preferentially reside near residues without α‐helical and β‐sheet secondary structures. These buried waters most often form primary hydrogen bonds to main‐chain atoms not involved in intramolecular hydrogen bonds, providing strong evidence that hydrating main‐chain atoms is a key structural role of buried water molecules. Additionally, the average B‐factor of protein atoms hydrogen‐bonded to waters is smaller than that of protein atoms forming intramolecular hydrogen bonds, and the average B‐factor of water molecules involved in primary hydrogen bonds with main‐chain atoms is smaller than the average B‐factor of water molecules involved in secondary hydrogen bonds to protein atoms that form concurrent intramolecular hydrogen bonds. To study the structural coupling between internal waters and buried polar atoms in detail we simulated the dynamics of wild‐type FKBP12, in which a buried water, Wat137, forms one side‐chain and multiple main‐chain hydrogen bonds. We mutated E60, whose side‐chain hydrogen bonds with Wat137, to Q, N, S or A, to modulate the multiplicity and geometry of hydrogen bonds to the water. Mutating E60 to a residue that is unable to form a hydrogen bond with Wat137 results in reorientation of the water molecule and leads to a structural readjustment of residues that are both near and distant to the water. We predict that the E60A mutation will result in a significantly reduced affinity of FKBP12 for its ligand FK506. The propensity of internal waters to hydrogen bond to buried polar atoms suggests that ordered water molecules may constitute fundamental structural components of proteins, particularly in regions where α‐helical or β‐sheet secondary structure is not present. Proteins 2005.

A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm

BackgroundIntrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus.ResultsWe describe the construction and validation of a large synthetic human single chain antibody fragment library based on a unique framework and optimized for cytoplasmic expression. Focusing the library by mimicking the natural diversity of CDR3 loops ensured that the scFvs were fully human and functional. We show that the library is highly diverse and functional since it has been possible to isolate by phage-display several strong binders against the five proteins tested in this study, the Syk and Aurora-A protein kinases, the αβ tubulin dimer, the papillomavirus E6 protein and the core histones. Some of the selected scFvs are expressed at an exceptional high level in the bacterial cytoplasm, allowing the purification of 1 mg of active scFv from only 20 ml of culture. Finally, we show that after three rounds of selection against core histones, more than half of the selected scFvs were active when expressed in vivo in human cells since they were essentially localized in the nucleus.ConclusionThis new library is a promising tool not only for an easy and large-scale selection of functional intrabodies but also for the isolation of highly expressed scFvs that could be used in numerous biotechnological and therapeutic applications.

Directing noble metal ion chemistry within a designed ferritin protein

Human H ferritin (HuHF) assembles from 24 four-helix bundles to form an approximately 500 kDa protein with an 8 nm internal cavity. HuHF provides a useful model for studying the transport of metal ions in solution to buried reaction sites in proteins. In this study, HuHF was redesigned to facilitate noble metal ion (Au(3+), Ag(+)) binding, reduction, and nanoparticle formation within the cavity. Computationally determined amino acid substitutions were targeted at four external and four internal surface sites. A variant with a total of 96 cysteines and histidines removed from the exterior surface and 96 non-native cysteines added to the interior surface retained wild-type stability and structure, as confirmed by X-ray crystallography, and promoted the formation of silver or gold nanoparticles within the protein cavity. Crystallographic studies with HuHF variants provide insight into how ferritins control access of metal ions to interior residues that perform chemistry.

An induced fit mechanism regulates p53 DNA binding kinetics to confer sequence specificity.

The p53 tumour suppressor gene, the most frequently mutated gene in human cancer, encodes a transcription factor that contains sequence‐specific DNA binding and homo‐tetramerization domains. Interestingly, the affinities of p53 for specific and non‐specific DNA sites differ by only one order of magnitude, making it hard to understand how this protein recognizes its specific DNA targets in vivo. We describe here the structure of a p53 polypeptide containing both the DNA binding and oligomerization domains in complex with DNA. The structure reveals that sequence‐specific DNA binding proceeds via an induced fit mechanism that involves a conformational switch in loop L1 of the p53 DNA binding domain. Analysis of loop L1 mutants demonstrated that the conformational switch allows DNA binding off‐rates to be regulated independently of affinities. These results may explain the universal prevalence of conformational switching in sequence‐specific DNA binding proteins and suggest that proteins like p53 rely more on differences in binding off‐rates, than on differences in affinities, to recognize their specific DNA sites.

Combinatorial protein design

Combinatorial protein libraries permit the examination of a wide range of sequences. Such methods are being used for denovo design and to investigate the determinants of protein folding. The exponentially large number of possible sequences, however, necessitates restrictions on the diversity of sequences in a combinatorial library. Recently, progress has been made in developing theoretical tools to bias and characterize the ensemble of sequences that fold into a given structure - tools that can be applied to the design and interpretation of combinatorial experiments.