Semisynthesis of a glycosylated Im7 analogue for protein folding studies

To establish a system to address questions concerning the influence of glycosylation on protein folding pathways, we have developed a semisynthetic route toward the immunity protein Im7. This fourhelix protein has been used extensively as model protein for folding studies. Native chemical ligation (NCL) affords an N-linked chitobiose glycoprotein analogue of Im7 with an Ala29Cys mutation. The semisynthetic approach relies on the solid-phase peptide synthesis (SPPS) of N-terminal thioesters (including helix I), in glycosylated or unglycosylated form, in combination with the expression of the C-terminal fragment of Im7 (containing helices II-IV). Detailed kinetic and thermodynamic analysis of the protein folding behavior reveals that semisynthetic Im7 analogues are well suited for protein folding studies and that the folding mechanism of the glycoprotein of this Im7 variant is not significantly altered over the unglycosylated analogue.     

The acid catalyzed cyclization of citral

Fourteen compounds have been identified as products of the acid catalyzed cyclization of citral under mild aqueous acid conditions. Three major products of the reaction, 1,8-epoxy-p-menth-2-ene (11) and trans- and cis-p-menth-2-ene-1,8-diol (16 and 18) are reported here for the first time as products from citral. Possible mechanistic pathways leading to the products are discussed.     

First nuclear moment measurement with radioactive beams by the recoil-in-vacuum technique: the g factor of the 2+1 state in 132Te

Following Coulomb excitation of the radioactive ion beam (RIB) 132Te at HRIBF we report the first use of the recoil-in-vacuum (RIV) method to determine the g factor of the 2(+)(1) state: g(973.9 keV 2(+) 132Te) = (+)0.35(5). The advantages offered by the RIV method in the context of RIBs and modern detector arrays are discussed.     

Decays of millisecond isomers in odd-oddN=81 nuclei146Tb,148Ho and150Tm

Following reactions induced by 245 MeV⁶⁰Ni ions, isomers have been identified in the odd-oddN=81 isotones¹⁴⁶Tb,¹⁴⁸Ho and¹⁵⁰Tm with half-lives of 1.18(2), 2.35(4) and 5.2(3) ms, respectively. Their decays have been characterized byγ-ray spectroscopy. The isomers are interpreted as (πh _11/2 νh ₁ ^2/?1 ) 10⁺ states decaying to members ofπh _11/2 νd ₃ ^2/?1 andπh ₁₁ ^2/?1 multiplets. The observed level spectra and M1 branching ratios are found to be in good agreement with shell model predictions based on empirical and Schiffer-Trueπν ^?1 residual interactions.     

An orthogonal design of experiment/exploratory data analysis for plutonium contamination

This paper describes a statistical-experimental design approach for identifying sources of Pu contamination in the radioactivity material analysis of dried shellfish at the National Institute of Standards and Technology (NIST). Identification and elimination of sources of contamination were required for certifications of the radioactivity concentration of actinides in the proposed reference material. Sources of contamination included the fume hood, glassware, and reagents. The experimental design employed reduced number of required experiments by a factor of 100. The result is an orthogonal design of experiment (ODEX) and subsequent data was analyzed using Exploratory Date Analysis (EDA), which narrowed the 200+ experiments to a manageable 16. This design made it possible to identify the sources and interactions of sources of contamination in a timely and cost effective manner.     

HDX-ESI-MS reveals enhanced conformational dynamics of the amyloidogenic protein β 2-microglobulin upon release from the MHC-1

The light chain of the major histocompatibility complex class 1 (MHC-1), the protein β ₂-microglobulin (β ₂m), has amyloidogenic properties that arise only upon its dissociation from the MHC-1. Here hydrogen/deuterium exchange electrospray ionization mass spectrometry (HDX-ESI-MS) has been used to compare the solution dynamics of β ₂m in its MHC-1 bound state compared with those of β ₂m as a free monomer. The capability of tandem mass spectrometry to dissociate the MHC-1 into its individual constituents in the gas phase following deuterium incorporation in solution has permitted the direct observation of the exchange properties of MHC-1 bound β ₂m for the first time. The HDX-ESI-MS data show clearly that the H→D exchange of MHC-1 bound β ₂m follows EX2 kinetics and that about 20 protons remain protected from exchange after 17 days. Free from the MHC-1, monomeric β ₂m exhibits significantly different HDX behavior, which encompasses both EX1 and EX2 kinetics. The EX2 kinetics indicate a tenfold increase in the rate of exchange compared with MHC-1 bound β ₂m, with just 10 protons remaining protected from EX2 exchange and therefore exchanging only via the EX1 mechanism. The EX1 kinetics observed for unbound β ₂m are consistent with unfolding of its exchange-protected core with a t_1/2 of 68 min (pH 7, 37° C). Thus, upon dissociation from the stabilizing influence of the MHC-1, free β ₂m becomes highly dynamic and undergoes unfolding transitions that result in an aggregation-competent protein.