ChemInformVolume 45, Issue 6 Natural Products ChemInform Abstract: A Bis(phosphine)-Modified Peptide Ligand for Stable and Luminescent Quantum Dots in Aqueous Media. Michael E. Jung, Michael E. Jung Dep. Chem. Biochem., Univ. Calif., Los Angeles, CA 90095, USASearch for more papers by this authorMichael Trzoss, Michael Trzoss Dep. Chem. Biochem., Univ. Calif., Los Angeles, CA 90095, USASearch for more papers by this authorJames M. Tsay, James M. Tsay Dep. Chem. Biochem., Univ. Calif., Los Angeles, CA 90095, USASearch for more papers by this authorShimon Weiss, Shimon Weiss Dep. Chem. Biochem., Univ. Calif., Los Angeles, CA 90095, USASearch for more papers by this author Michael E. Jung, Michael E. Jung Dep. Chem. Biochem., Univ. Calif., Los Angeles, CA 90095, USASearch for more papers by this authorMichael Trzoss, Michael Trzoss Dep. Chem. Biochem., Univ. Calif., Los Angeles, CA 90095, USASearch for more papers by this authorJames M. Tsay, James M. Tsay Dep. Chem. Biochem., Univ. Calif., Los Angeles, CA 90095, USASearch for more papers by this authorShimon Weiss, Shimon Weiss Dep. Chem. Biochem., Univ. Calif., Los Angeles, CA 90095, USASearch for more papers by this author First published: 23 January 2014 https://doi.org/10.1002/chin.201406215Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume45, Issue6February 11, 2014 RelatedInformation

We describe a new class of ligands for semiconductor nanoparticles (quantum dots = QDs), which bind well and allow for their facile dissolution in aqueous solution. As a proof of principle, we have designed and synthesized a novel bis(phosphine)-modified peptide (BPMP) and shown that it has the ability to solubilize quantum dots in aqueous media. We further showed that the corresponding phosphine oxide derivatives of these new ligands are less good at solubilizing the quantum dots. These new bis(phosphine)-modified peptide ligands are easy to prepare and may well replace thiol-containing binding sequences in functionalized peptides for quantum dot coating, potentially resulting in quantum dots with higher quantum yields.

Many double-stranded DNA viruses employ ATP-driven motors to translocate their genomes into small, preformed viral capsids against large forces resisting confinement. Here, we show via direct single-molecule measurements that a mutation T194M downstream of the Walker B motif in the phage λ gpA packaging motor causes an 8-fold reduction in translocation velocity without substantially changing processivity or force dependence, whereas the mutation G212S in the putative C (coupling) motif causes a 3-fold reduction in velocity and a 6-fold reduction in processivity. Meanwhile a T194M pseudorevertant (T194V) showed a near restoration of the wild-type dynamics. Structural comparisons and modeling show that these mutations are in a loop-helix-loop region that positions the key residues of the catalytic motifs, Walker B and C, in the ATPase center and is structurally homologous with analogous regions in chromosome transporters and SF2 RNA helicases. Together with recently published studies of SpoIIIE chromosome transporter and Ded1 RNA helicase mutants, these findings suggest the presence of a structurally conserved region that may be a part of the mechanism that determines motor velocity and processivity in several different types of nucleic acid translocases.

dsDNA phages and viruses employ DNA packaging motors to translocate their genomes into small viral capsids against enormous internal pressures. Structural data, models, and sequence alignments have revealed the homology of critical putative functional domains of various nucleic acid translocases, including viral packaging motors, RNA helicases, and chromosome transporters. We used optical tweezers and mutational analysis to explore which functional domains of viral packaging motors govern their force generation and determine the velocities of packaging. A Q motif mutant of the phage λ DNA packaging motor, Y46F, was shown to have a decreased velocity (∼40% less than WT), increased slipping (∼10X WT), and steeper force-velocity dependence (∼6X WT), showing that the Q motif governs the force generation in translocation and DNA-motor interactions. In addition, we show that mutants with residue changes located in a previously undetermined domain of the motor, T194M and G212S, package dsDNA into viral capsids at ∼8X and ∼ 3X slower velocities than wild type (WT), respectively. Meanwhile a T194M pseudo-revertant (T194V) showed a near restoration of the WT velocity. The single molecule measurements of motor mutant translocation dynamics, genetic screening experiments, and structural modeling of ring ATPase dsDNA translocases suggest the location of a "velocity controller" domain within the phage λ packaging motor downstream the putative Walker B motif, which might be generalizable to other ring ATPase nucleic acid translocases. Importantly, this evidence may aid in explaining the different packaging rates of various dsDNA phages.

A key step in the assembly of many viruses is the packaging of double-stranded DNA into a procapsid shell by the action of an ATP-powered molecular motor. We use optical tweezers to measure the packaging of single DNA molecules into single viral proheads in real time. We can measure DNA binding and initiation of translocation, DNA translocation dynamics, force generated by the motor, and can infer the forces resisting DNA confinement. We have developed approaches to study three different viruses: Bacteriophages phi29, lambda, and T4. These viruses have different capsid sizes and shapes, genome lengths, and structural and biochemical differences in their packaging motors, resulting in differing DNA packaging dynamics. All three motors translocate DNA processively and generate high forces exceeding 50 piconewtons, but the motor velocities vary 10-fold. In the lambda system we have found evidence for an effect of procapsid expansion on the packaging dynamics and evidence for force-induced capsid rupture in the absence of a putative stabilizing protein. We are currently investigating motor structure-function relationships by analyzing effects of point mutations. Amongst the mutants we have identified are one that exhibits a motor velocity roughly one-tenth that of the wild type, and one that exhibits increased pausing and slipping. These studies shed light on the various functional domains of viral packaging motors.

A key step in the assembly of many viruses is the packaging of DNA into preformed procapsids by an ATP-powered molecular motor. To shed light on the motor mechanism we used single-molecule optical tweezers measurements to study the effect of mutations in the large terminase subunit in bacteriophage λ on packaging motor dynamics. A mutation, K84A, in the putative ATPase domain driving DNA translocation was found to decrease motor velocity by ≈40% but did not change the force dependence or decrease processivity substantially. These findings support the hypothesis that a deviant “Walker A-like” phosphate-binding motif lies adjacent to residue 84. Another mutation, Y46F, was also found to decrease motor velocity by ≈40% but also increase slipping during DNA translocation by >10-fold. These findings support the hypothesis that viral DNA packaging motors contain an adenine-binding motif that regulates ATP hydrolysis and substrate affinity analogous to the “Q motif” recently identified in DEAD-box RNA helicases. We also find impaired force generation for the Y46F mutant, which shows that the Q motif plays an important role in determining the power and efficiency of the packaging motor.

Optical tweezers measurements were employed to directly observe viral DNA packaging in wild type and packaging mutants of bacteriophage lambda. Several key findings are reported here: DNA packaging by purified wild type lambda motors was measured for the first time, showing nearly identical behavior in packaging DNA to crude extracts of terminase components. A slow packaging lambda mutant, T194M, was found to package DNA at similar to 10x slower velocity than wild type. Meanwhile another packaging mutant Y46F was found to package DNA slower than the wild type (60-70% the velocity of the wild type velocity) as well as slipping >10x more frequently (per length of DNA) than wild type. Another mutant (K84A) showed slower packaging (60-70% the velocity of wildtype), but displayed slipping and pausing behavior similar to wild type. Finally the pausing and slipping dependence on length of DNA packaged of the various terminases studied was discovered, suggesting further structural defects of the mutants that are detrimental to translocation. These studies confirm the location of an ATPase center in the N-terminal portion of gpA which is responsible for translocation of dsDNA.

Since their first appearance as optical probes in biological imaging [1, 2], qdots have been applied in most biotechnological applications using fluorescence, including DNA array technology, immunofluorescence assays (reviewed in [3]), and cell and animal biology (reviewed in [4, 5]. Qdots have gained wide acceptance by the scientific community and the biotechnology industry as new fluorescent, nonisotopic labels of unmatched potentials. Most appealing to the biosciences are the high brightness, high resistance to photobleaching, and the ability to size-tune fluorescent emission of these inorganic-biological hybrid nanostructures. Qdots have also proved to be excellent probes for two-photon confocal microscopy and deep-tissue imaging given their large two-photon absorption cross-section [6–9]. But it is when combined with ultrasensitive microscopy techniques that qdots have shown their true potential by allowing visualization of cellular processes down to the molecular scale [10–13]. The enhanced photophysical properties of qdots can fulfill the stringent requirements needed to provide spatial, temporal, and structural information at all length scales: from the whole body down to the nanometer resolution with a single probe. Fluorescent nanocrystals of semiconductor material, which are generally synthesized in solutions of nonpolar organic solvents using colloidal chemistry [14], require additional chemical modifications to be solubilized in aqueous buffers and functionalized for biological applications (see Chapter 1, “Colloidal Quantum Dots: Synthesis, Photophysical Properties, and Biofunctionalization Strategies,” by Susumu et al. for an in-depth discussion). This can be achieved by either surfactant exchange, a process primarily driven by mass-action in which the native TOP/TOPO hydrophobic surface ligands are substituted with bifunctional amphiphilic ones, or by insulation of the original hydrophobic qdot within a heterofunctional amphiphilic coating [4, 5]. These various qdot solubilization strategies have included the following: (1) ligand exchange with small thiol-containing

On the dot: A genetic-engineering approach to synthesizing a rationally designed recombinant peptide (termed rFC3) in Escherichia coli can be used for the solubilization of quantum dots (QDs). The rFC3 sequence contains only natural amino acids. Exchange of this peptide with CdSe–ZnS QDs coated with hydrophobic solvent allows surface recognition of the peptide (see picture). Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2296/2007/z600516_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Peptide-coated quantum dot-photosensitizer conjugates were developed using novel covalent conjugation strategies on peptides which overcoat quantum dots (QDs). Rose bengal and chlorin e6, photosensitizers (PSs) that generate singlet oxygen in high yield, were covalently attached to phytochelatin-related peptides. The photosensitizer-peptide conjugates were subsequently used to overcoat green- and red-emitting CdSe/CdS/ZnS nanocrystals. Generation of singlet oxygen could be achieved via indirect excitation through Förster (fluorescence) resonance energy transfer (FRET) from the nanocrystals to PSs, or by direct excitation of the PSs. In the latter case, by using two color excitations, the conjugate could be simultaneously used for fluorescence imaging and singlet oxygen generation. Singlet oxygen quantum yields as high as 0.31 were achieved using 532-nm excitation wavelengths.

After much effort in surface chemistry development and optimization by several groups, fluorescent semiconductor nanocrystals probes, also known as quantum dots or qdots, are now entering the realm of biological applications with much to offer to biologists. The road to success has been paved with hurdles but from these efforts has stemmed a multitude of original surface chemistries that scientists in the biological fields can draw from for their specific biological applications. The ability to easily modulate the chemical nature of qdot surfaces by employing one or more of the recently developed qdot coatings, together with their exceptional photophysics have been key elements for qdots to acquire a status of revolutionary fluorescent bio-probes. Indeed, the unique properties of qdots not only give biologists the opportunity to explore advanced imaging techniques such as single molecule or lifetime imaging but also to revisit traditional fluorescence imaging methodologies and extract yet unobserved or inaccessible information in vitro or in vivo.

Quantum dots (QDOTs) have been widely recognized by the scientific community and the biotechnology industry, as witnessed by the exponential growth of this field in the past several years. We describe the synthesis and characterization of visible and near infrared QDots-a critical step for engineering organic molecules like proteins and peptides for building nanocomposite materials with multifunctional properties suitable for biological applications

CdSe/CdS/ZnS nanorods (NRs) of three aspect ratios were coated with phytochelatin-related peptides and studied using fluorescence correlation spectroscopy (FCS). Theoretical predictions of the NRs' rotational diffusion contribution to the correlation curves were experimentally confirmed. We monitored rotational and translational diffusion of NRs and extracted hydrodynamic radii from the extracted diffusion constants. Translational and rotational diffusion constants (D(trans) and D(rot)) for NRs were in good agreement with Tirado and Garcia de la Torre's as well as with Broersma's theories when accounting for the ligand dimensions. NRs fall in the size range where rotational diffusion can be monitored with higher sensitivity than translational diffusion due to a steeper length dependence, D(rot) approximately L(-)(3) versus D(trans) approximately L(-)(1). By titrating peptide-coated NRs with bovine serum albumin, we monitored (nonspecific) binding through rotational diffusion and showed that D(rot) is an advantageous observable for monitoring binding. Monitoring rotational diffusion of bioconjugated NRs using FCS might prove to be useful for observing binding and conformational dynamics in biological systems.

We have synthesized high quality type-II CdTe/CdSe near infrared quantum dots using successive ion layer adsorption and reaction chemistry. Transmission electron microscopy reveals that CdTe/CdSe can be synthesized layer by layer yielding quantum dots of narrow size distribution. Excitation and photoluminescence spectra reveal discrete type-II transitions, which correspond to energy lower that type-I bandgap. We have used a peptide coating technique on type-II and commercial near infrared quantum dots for delivery in live animals and cultured cells.

We have developed a new functionalization approach for semiconductor nanocrystals based on a single-step exchange of surface ligands with custom-designed peptides. This peptide-coating technique yield small, monodisperse and very stable water-soluble NCs that remain bright and photostable. We have used this approach on several types of core and core-shell NCs in the visible and near-infrared spectrum range and used fluorescence correlation spectroscopy for rapid assessment of the colloidal and photophysical properties of the resulting particles. This peptide coating strategy has several advantages: it yields probes that are immediately biocompatible; it is amenable to improvements of the different properties (solubilization, functionalization, etc) via rational design, parallel synthesis, or molecular evolution; it permits the combination of several functions on individual NCs. These functionalized NCs have been used for diverse biomedical applications. Two are discussed here: single-particle tracking of membrane receptor in live cells and combined fluorescence and PET imaging of targeted delivery in live animals.