Dynein, a homodimeric protein complex, plays a pivotal role in retrograde transportation along microtubules within cells. It consists of various subunits, among which the light intermediate chain (LIC) performs diverse functions, including cargo adaptor binding. In contrast to the vertebrate LIC homolog LIC1, LIC2 has received relatively limited characterization thus far, despite partially orthogonal functional roles. In this study, we present a near-to-complete backbone NMR chemical shift assignment of the C-terminal region of the light intermediate chain 2 of human dynein 1 (LIC2-C). We perform a comparative analysis of the secondary structure propensity of LIC2-C with the one previously reported for LIC1-C and show that the two transient helices in LIC1 that interact with motor adaptors are also present in LIC2.

The accurate analysis of continuous-wave electron spin resonance (cw ESR) spectra of biological or organic free-radicals and paramagnetic metal complexes is key to understanding their structure-function relationships and electrochemical properties. The current methods of analysis based on simulations often fail to extract the spectral information accurately. In addition, such analyses are highly sensitive to spectral resolution and artifacts, users' defined input parameters and spectral complexity. We introduce a simulation-independent spectral analysis approach that enables broader application of ESR. We use a wavelet packet transform-based method for extracting g values and hyperfine (A) constants directly from cw ESR spectra. We show that our method overcomes the challenges associated with simulation-based methods for analyzing poorly/partially resolved and unresolved spectra, which is common in most cases. The accuracy and consistency of the method are demonstrated on a series of experimental spectra of organic radicals and copper-nitrogen complexes. We showed that for a two-component system, the method identifies their individual spectral features even at a relative concentration of 5% for the minor component.

Protein methyl groups can participate in multiple motional modes on different time scales. Sub-nanosecond to nano-second time scale motions of methyl axes are particularly challenging to detect for small proteins in solutions. In this work we employ NMR relaxation interference between the methyl H-H/H-C dipole-dipole interactions [Sun&Tugarinov, J. Magn. Reason. 2012] to characterize methyl axes motions as a function of temperature in a small model protein villin headpiece subdomain (HP36), in which all non-exchangeable protons are deuterated with the exception of methyl groups of leucine and valine residues. The data points to the existence of slow motional modes of methyl axes on sub-nanosecond to nanosecond time scales. Further, at high temperatures for which the overall tumbling of the protein is on the order of 2 ns, we observe a coupling between the slow internal motion and the overall molecular tumbling, based on the anomalous order parameters and their temperature-dependent trends. The addition of 28%(w/w) glycerol-d8 increases the viscosity of the solvent and separates the timescales of internal and overall tumbling, thus permitting for another view of the necessity of the coupling assumption for these sites at high temperatures.

The objective of spectral analysis is to resolve and extract relevant features from experimental data in an optimal fashion. In continuous-wave (cw) electron spin resonance (ESR) spectroscopy, both values of a paramagnetic center and hyperfine splitting caused by its interaction with neighboring magnetic nuclei in a molecule provide important structural and electronic information. However, in the presence of - and/or -anisotropy and/or large number of resonance lines, spectral analysis becomes highly challenging. Either high-resolution experimental techniques are employed to resolve the spectra in those cases or a range of suitable ESR frequencies are used in combination with simulations to identify the corresponding and values. In this work, we present a wavelet transform technique in resolving both simulated and experimental cW-ESR spectra by separating the hyperfine and super-hyperfine components. We exploit the multiresolution property of wavelet transforms that allow the separation of distinct features of a spectrum based on simultaneous analysis of spectrum and its varying frequency. We retain the wavelet components that stored the hyperfine and/or super-hyperfine features, while eliminating the wavelet components representing the remaining spectrum. We tested the method on simulated cases of metal-ligand adducts at L-, S-, and X-band frequencies, and showed that extracted values, hyperfine and super-hyperfine coupling constants from simulated spectra, were in excellent agreement with the values of those parameters used in the simulations. For the experimental case of a copper(II) complex with distorted octahedral geometry, the method was able to extract and hyperfine coupling constant values, and revealed features that were buried in the overlapped spectra.

Over the years, various techniques have been utilized to study the function and phenotype of antigen-binding B cells in the primary repertoire following immunization, infection, and development of autoimmunity. Due to the low frequency of antigen-reactive B cells (<0.05% of lymphocytes) in the periphery, preliminary enrichment of cells is necessary to achieve sufficient numbers for statistically sound characterization, especially when downstream analytic platform use, e.g., CyTOF, is low throughput. We previously described a method to detect and enrich antigen-reactive B cells from peripheral blood and tissues using biotinylated antigens in conjunction with magnetic nanoparticles, preparative to a downstream analysis by ELISPOT and flow cytometry. While mass cytometry (CyTOF) enables high dimensional immunophenotyping of over 40 unique parameters on a single-cell level, its low throughput compared to flow cytometry and requirement for removal of metal contaminants, such as nanoparticles, made it particularly unsuitable for studies of rare cells in a mixed population. Here we describe a novel CyTOF-compatible approach for multiplexed enrichment of antigen-reactive B cells, e.g., insulin and tetanus toxoid, using cleavable magnetic nanoparticles. This method allows improved monitoring of the phenotype and function of antigen-reactive B cells during the development of disease or after immunization while minimizing the amount of sample and run times needed.

Studying the correlation between temperature-driven molecular structure and nuclear spin dynamics is essential to understanding fundamental design principles for thermometric nuclear magnetic resonance spin-based probes. Herein, we study the impact of progressively encapsulating ligands on temperature-dependent Co (spin-lattice) and (spin-spin) relaxation times in a set of Co(III) complexes: K[Co(CN)] (); [Co(NH)]Cl (); [Co(en)]Cl (), en = ethylenediamine); [Co(tn)]Cl (), tn = trimethylenediamine); [Co(tame)]Cl (), tame = triaminomethylethane); and [Co(dinosar)]Cl (), dinosar = dinitrosarcophagine). Measurements indicate that Co and increase with temperature for - between 10 and 60 °C, with the greatest Δ /Δ and Δ /Δ temperature sensitivities found for and , 5.3(3)% /°C and 6(1)% /°C, respectively. Temperature-dependent * (dephasing time) analyses were also made, revealing the highest Δ */Δ sensitivities in structures of greatest encapsulation, as high as 4.64% */°C for . Calculations of the temperature-dependent quadrupolar coupling parameter, Δ /Δ enable insight into the origins of the relative Δ /Δ values. These results suggest tunable quadrupolar coupling interactions as novel design principles for enhancing temperature sensitivity in nuclear spin-based probes.

We present a strategy for stereospecific NMR assignment of H and H protons in mid-size proteins (~150 residues). For such proteins, resonance overlap in standard experiments is severe, thereby preventing unambiguous assignment of a large fraction of β-methylenes. To alleviate this limitation, assignment experiments may be run in high static fields, where higher decoupling power is required. Three-bond H-H J-couplings ( ) are critical for stereospecific assignments of β-methylene protons, and for determining rotameric χ states. Therefore, we modified a pulse sequence designed to measure accurate couplings such that probe heating was reduced, while the decoupling performance was improved. To further increase the resolution, we applied non-uniform sampling (NUS) schemes in the indirect H and C dimensions. The approach was applied to two medium-sized proteins, odorant binding protein 22 (OBP22; 14.4 kDa) and Pin1 (18.2 kDa), at 900 MHz polarizing fields. The coupling values obtained from NUS and linear sampling were extremely well correlated. However, NUS decreased the overlap of H protons, thus supplying a higher yield of extracted coupling values when compared with linear sampling. A similar effect could be achieved with linear prediction applied to the linearly sampled data prior to the Fourier transformation. Finally, we used couplings from Pin1 in combination with either conventional or exact nuclear Overhauser enhancement (eNOE) restraints to determine the stereospecific assignments of β-methylene protons. The use of eNOEs further increased the fraction of unambiguously assigned resonances when compared with procedures using conventional NOEs.

Bleomycins are antitumor antibiotics that can chelate a metal center and cause site-specific DNA cleavage at 5'-Gpyrimidine-3' regions of DNA. These antibiotics are successful in the treatment of various cancers, but are known to cause pulmonary fibrosis to patients under bleomycin regimes. Substantial research has resulted in the development of over 300 bleomycin analogs, aiming to improve the therapeutic index of the drug. Previous studies have proposed that the lung toxicity caused by bleomycin is related to the C-terminal regions of these drugs, which have been shown to closely interact with DNA in metal-bleomycin-DNA complexes. Some of the research studying metallo-bleomycin-DNA interactions have suggested three different binding modes of the metal form of the drug to DNA, including total and/or partial intercalation, and minor groove binding. However, there is still lack of consensus regarding this matter, and solid conclusions on the subject have not yet been established. Previously we investigated the diverse levels of disruption caused to DNA hairpins containing 5'-GC-3' and 5'-GT-3' binding sites, which are consequence of the binding of bleomycins with different C-termini. The results of these investigation indicate that both the DNA-binding site and the bleomycin C-termini have an impact on the final conformations of drug and target. The present study focuses on the structural alterations exhibited by Zn(II)bleomycin-A, -B, -A and Zn(II)peplomycin upon binding to DNA hairpins containing 5'-GC-3' and 5'-GT-3' binding sites. Evidence that each Zn(II)bleomycin is structurally affected depending on both its C-terminus and the DNA-binding site present in the hairpin is provided.