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Glycoconjugate vaccines are important additions to the existing means for prevention of diseases caused by bacterial and viral pathogens. Conjugating carbohydrates to proteins is a crucial step in the development of these vaccines. Traditional mass spectrometry techniques, such as MALDI-TOF and SELDI-TOF, have difficulties in detecting glycoconjugates with high molecular masses. Mass photometry (MP) is a single-molecule technique that has been recently developed, which allows mass measurements of individual molecules and generates mass distributions based on hundreds to thousands of these measurements. In this study, we evaluated the performance of MP in monitoring carbohydrate-protein conjugation reactions and characterization of conjugates. Three different glycoconjugates were prepared from carrier protein BSA, and one from a large protein complex, a virus capsid with 3.74 MDa molecular mass. The masses measured by MP were consistent with those obtained by SELDI-TOF-MS and SEC-MALS. The conjugation of BSA dimer to carbohydrate antigen was also successfully characterized. This study shows that the MP technique is a promising alternative to methods developed earlier for monitoring glycoconjugation reactions and characterization of glycoconjugates. It measures intact molecules in solution and it is highly accurate over a wide mass range. MP requires only a very small amount of sample and has no specific buffer constraints. Other MP advantages include minimal cost of consumables and rapid data collection and analysis. Its advantages over other methods make it a valuable tool for researchers in the glycoconjugation field. [ABSTRACT FROM AUTHOR]

Mass photometry (MP) is a relatively new experimental technique with a quickly expanding list of applications. Using optical detection, MP measures the mass of individual molecules to obtain molecular mass distributions of proteins and other biomolecules in solution. The combination of speed, sensitivity, and very low sample consumption with label- and immobilization-free detection sets MP apart from other analytical methods. An increasing number of laboratories incorporates mass photometry as a routine sample analysis technique. However, MP measurements can sometimes be challenging, especially for users without previous experience with single-molecule techniques. Here, we present a protocol for the determination of protein molecular mass distributions by MP. It describes the sample and materials preparation as well as data collection and analysis. The advantages and limitations of this technique and the potential sources of artifacts are also given. This protocol can be used by new MP users and serve as a checklist for laboratories routinely performing MP experiments to guide consistent data collection and documentation. [ABSTRACT FROM AUTHOR]

A small-scale ITC benchmarking study was performed involving 9 biophysics laboratories/facilities, to evaluate inter-laboratory and intra-laboratory basal levels of uncertainty. Our prime goal was to assess a number of important factors that can influence both the data gathered by this technique and the thermodynamic parameter values derived therefrom. In its first part, the study involved 5 laboratories and 13 different instruments, working with centrally prepared samples and the same experimental protocol. The second part involved 4 additional laboratories and 6 more instruments, where the users prepared their own samples according to provided instructions and did the experiments following the same protocol as in the first part. The study design comprised: (1) selecting a minimal set of laboratories; (2) providing very stable samples; (3) providing samples not requiring preparation or manipulation; and (4) providing a well-defined and detailed experimental protocol. Thus, we were able to assess: (i) the variability due to instrument and data analysis performed by each user on centrally prepared samples; (ii) the comparability of data retrieved when using 4 different software packages to analyze the same data, besides the data analysis carried out by the different users on their own experimental results; and (iii) the variability due to local sample preparation (second part of the study). Individual values, as well as averages and standard deviations for the binding parameters for EDTA-cation interaction, were used as metrics for comparing the equilibrium association constant (logK), enthalpy of interaction (ΔH), and the so-called "stoichiometry" (n), a concentration-correction factor. [ABSTRACT FROM AUTHOR]

Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they lose their fluorescence after heavy fixation, hindering applications such as correlative light and electron microscopy (CLEM). Here we report engineered variants of the photoconvertible Eos fluorescent protein that fluoresce and photoconvert normally in heavily fixed (0.5-1% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy. [ABSTRACT FROM AUTHOR]

Tubulin tyrosine ligase (TTL) catalyzes the post-translational C-terminal tyrosination of ?-tubulin. Tyrosination regulates recruitment of microtubule-interacting proteins. TTL is essential. Its loss causes morphogenic abnormalities and is associated with cancers of poor prognosis. We present the first crystal structure of TTL (from Xenopus tropicalis), defining the structural scaffold upon which the diverse TTL-like family of tubulin-modifying enzymes is built. TTL recognizes tubulin using a bipartite strategy. It engages the tubulin tail through low-affinity, high-specificity interactions, and co-opts what is otherwise a homo-oligomerization interface in structurally related ATP grasp-fold enzymes to form a tight hetero-oligomeric complex with the tubulin body. Small-angle X-ray scattering and functional analyses reveal that TTL forms an elongated complex with the tubulin dimer and prevents its incorporation into microtubules by capping the tubulin longitudinal interface, possibly modulating the partition of tubulin between monomeric and polymeric forms. [ABSTRACT FROM AUTHOR]

Fluorescent probes bound to DNA typically display nanosecond decay times and reveal only nanosecond motions. We extend the time range of measurable DNA dynamics using [Ru(bpy)2(dppz)]2+ (bpy=2,2’-bipyridine, dppz=dipyrido[3,2-a:2’,3’-c]phenazine) (RuBD) which displays a mean lifetime near 90 ns. To test the usefulness of RuBD as a probe for diffusive processes in calf thymus DNA, we compared the efficiencies of fluorescence resonance energy transfer (FRET) using three donors which display lifetimes near 5 ns for acridine orange (AO), 22 ns for ethidium bromide (EB) and 92 ns for RuBD, with nile blue (NB) as the acceptor. The Förster distances for AO-NB, EB-NB and RuBD-NB donor-acceptor pairs were 42.3, 52.3, and 30.6 A, respectively. All three donors showed dramatic decreases in fluorescence intensities and more rapid intensity decays with increasing NB concentrations. The intensity decays of AO and EB in the presence of varying concentrations of NB were satisfactorily described by the one-dimensional FRET model without diffusion (Blumen and Manz, 1979). In the case of the long-lifetime donor RuBD, the experimental phase and modulation somewhat deviated from the recovered values computed from this model. The recovered NB concentrations and FRET efficiencies from the model were slightly larger than the expected values, however, the recovered and expected values did not show a significant difference. Thus, it is suggested that the lifetime of RuBD is too short to measure diffusive processes in calf thymus DNA. [ABSTRACT FROM AUTHOR]

We report observation of high quantum yield, long-lifetime fluorescence from a red dye BO-PRO-3 excited by resonance energy transfer (RET). The acceptor fluorescence was highly enhanced upon binding to the donor-labeled DNA. A ruthenium complex (Ru) was chosen as a donor in this system because of its long fluorescence lifetime. Both donor and acceptor were non-covalently bound to DNA. Emission from the donor-acceptor system (DA) at wavelengths exceeding 600 nm still preserves the long-lifetime component of the Ru donor, retaining average fluorescence lifetimes in the range of 30–50 ns. Despite the low quantum yield of the Ru donor in the absence of acceptor, its overall quantum yield of the DA pair was increased by energy transfer to the higher quantum yield acceptor BO-PRO-3. The wavelength-integrated intensity of donor and acceptor bound to DNA was many-fold greater than the intensity of the donor and acceptor separately bound to DNA. The origin of this effect is due to an efficient energy transfer from the donor, competing with non-radiative depopulation of the donor excited state. The distinctive features of DA complexes can be used in the development of a new class of engineered luminophores that display both long lifetime and long-wavelength emission. Similar DA complexes can be applied as proximity indicators, exhibiting strong fluorescence of adjacently located donors and acceptors over the relatively weak fluorescence of separated donors and acceptors. [ABSTRACT FROM AUTHOR]

We describe multiphoton excitation of the lanthanides europium (Eu3+) and terbium (Tb3+) when these ions are complexed with nucleic acids, proteins, and fluorescent chelators. In all cases excitation occurs by multiphoton absorption of the sensitizers. For the nucleotide GDP and an oligonucleotide with several guanines, the sensitized emission of Tb3+ excited at 776 nm indicated a three-photon process. For Tb3+ bound to the wild-type troponin C and a single tryptophan mutant (26W), excitation at 794 nm was also close to a three-photon process. For lanthanide chelators containing various sensitizers, we observed three-photon excitation in the case of methyl anthranilate, a mixuture of two- and three-photon excitation for carbostyril 124, and a two-photon process with a coumarin derivative. In the case of coumarin-sensitized emission of Eu3+ varied from a two- to a three-photon process at wavelengths ranging from 780 to 880 nm. The sensitized luminescence also shows significantly higher photostability compared to the fluorescence from the organic fluorophores alone. These results suggest the use of multiphoton-induced sensitized lanthanide fluorescence in biochemistry and cellular imaging. [ABSTRACT FROM AUTHOR]

Near infrared (NIR) multi-photon excitation of the NIR-emitting lanthanides neodymium (Nd3+) and ytterbium (Yb3+) sensitized by a fluorescein-linked chelator was demonstrated. Because tissues display minimal absorbance near the excitation wavelength of 800 nm, and because the lanthanides display long decay times, these results suggest the use of Nd3+ and Yb3+ as luminescent probes in tissues with multi-photon excitation. [ABSTRACT FROM AUTHOR]

A correction to the article "Fixation-resistant photoactivatable fluorescent proteins for CLEM," by Maria G. Paez-Segala, Mei G. Sun, and Sarada Viswanathan that was published online on January 12, 2015 is presented.