Efficient and economic protein labeling for NMR in mammalian expression systems: Application to a preT-cell and T-cell receptor protein.

Protein nuclear magnetic resonance (NMR) spectroscopy relies on the ability to isotopically label polypeptides, which is achieved through heterologous expression in various host organisms. Most commonly, Escherichia coli is employed by leveraging isotopically substituted ammonium and glucose to uniformly label proteins with 15N and 13C, respectively. Moreover, E. coli can grow and express proteins in uniformly deuterium-substituted water (D2O), a strategy useful for experiments targeting high molecular weight proteins. Unfortunately, many proteins, particularly those requiring specific posttranslational modifications like disulfide bonding or glycosylation for proper folding and/or function, cannot be readily expressed in their functional forms using E. coli-based expression systems. One such class of proteins includes T-cell receptors and their related preT-cell receptors. In this study, we present an expression system for isotopic labeling of proteins using a nonadherent human embryonic kidney cell line, Expi293F, and a specially designed media. We demonstrate the application of this platform to the ß subunit common to both receptors. In addition, we show that this expression system and media can be used to specifically label amino acids Phe, Ile, Val, and Leu in this system, utilizing an amino acid-specific labeling protocol that allows targeted incorporation at high efficiency without significant isotopic scrambling. We demonstrate that this system can also be used to express proteins with fluorinated amino acids. We were routinely able to obtain an NMR sample with a concentration of 200 µM from 30 mL of culture media, utilizing less than 20 mg of the labeled amino acids.

Selective methyl labeling of eukaryotic membrane proteins using cell-free expression.

Structural characterization of membrane proteins and other large proteins with NMR relies increasingly on perdeuteration combined with incorporation of specifically protonated amino acid moieties, such as methyl groups of isoleucines, valines, or leucines. The resulting proton dilution reduces dipolar broadening producing sharper resonance lines, ameliorates spectral crowding, and enables measuring of crucial distances between and to methyl groups. While incorporation of specific methyl labeling is now well established for bacterial expression using suitable precursors, corresponding methods are still lacking for cell-free expression, which is often the only choice for producing labeled eukaryotic membrane proteins in mg quantities. Here we show that we can express methyl-labeled human integral membrane proteins cost-effectively by cell-free expression based of crude hydrolyzed ILV-labeled OmpX inclusion bodies. These are obtained in Escherichia coli with very high quantity and represent an optimal intermediate to channel ILV precursors into the eukaryotic proteins.

DNA dynamics is likely to be a factor in the genomic nucleotide repeats expansions related to diseases.

Trinucleotide repeats sequences (TRS) represent a common type of genomic DNA motif whose expansion is associated with a large number of human diseases. The driving molecular mechanisms of the TRS ongoing dynamic expansion across generations and within tissues and its influence on genomic DNA functions are not well understood. Here we report results for a novel and notable collective breathing behavior of genomic DNA of tandem TRS, leading to propensity for large local DNA transient openings at physiological temperature. Our Langevin molecular dynamics (LMD) and Markov Chain Monte Carlo (MCMC) simulations demonstrate that the patterns of openings of various TRSs depend specifically on their length. The collective propensity for DNA strand separation of repeated sequences serves as a precursor for outsized intermediate bubble states independently of the G/C-content. We report that repeats have the potential to interfere with the binding of transcription factors to their consensus sequence by altered DNA breathing dynamics in proximity of the binding sites. These observations might influence ongoing attempts to use LMD and MCMC simulations for TRS-related modeling of genomic DNA functionality in elucidating the common denominators of the dynamic TRS expansion mutation with potential therapeutic applications.

A nonlinear dynamic model of DNA with a sequence-dependent stacking term.

No simple model exists that accurately describes the melting behavior and breathing dynamics of double-stranded DNA as a function of nucleotide sequence. This is especially true for homogenous and periodic DNA sequences, which exhibit large deviations in melting temperature from predictions made by additive thermodynamic contributions. Currently, no method exists for analysis of the DNA breathing dynamics of repeats and of highly G/C- or A/T-rich regions, even though such sequences are widespread in vertebrate genomes. Here, we extend the nonlinear Peyrard-Bishop-Dauxois (PBD) model of DNA to include a sequence-dependent stacking term, resulting in a model that can accurately describe the melting behavior of homogenous and periodic sequences. We collect melting data for several DNA oligos, and apply Monte Carlo simulations to establish force constants for the 10 dinucleotide steps (CG, CA, GC, AT, AG, AA, AC, TA, GG, TC). The experiments and numerical simulations confirm that the GG/CC dinucleotide stacking is remarkably unstable, compared with the stacking in GC/CG and CG/GC dinucleotide steps. The extended PBD model will facilitate thermodynamic and dynamic simulations of important genomic regions such as CpG islands and disease-related repeats.