Click‐Tag and Amine‐Tag: Chemical Tag Approaches for Efficient Protein Labeling In Vitro and on Live Cells using the Naturally Split Npu DnaE Intein

Protein labeling with synthetic moieties remains in many cases a technically challenging or unresolved task. Two new and simple concepts are presented. In both approaches, a very short tag of only a few amino acids is prepared with the desired chemical modification and, in a second step, it is transferred to the protein of interest by protein trans‐splicing. For the amine‐tag, a recombinant intein fragment free of lysine residues was generated such that the amine group of the N terminus could be selectively modified with regular amine‐reactive reagents. Thus, standard bioconjugation procedures without any chemical synthesis could be applied without modification of lysines in the protein of interest. For the click‐tag, protein trans‐splicing was combined with unnatural amino acid mutagenesis and subsequent bioorthogonal side chain modification, as demonstrated for click chemistry using p‐azidophenylalanine. By the two‐step strategy, exposure of the protein of interest to the copper catalyst was avoided.     

Photobodies: Light‐Activatable Single‐Domain Antibody Fragments

Photocaged antibody fragments, termed photobodies, have been developed that are impaired in their antigen‐binding capacity and can be activated by irradiation with UV light (365 nm). This rational design concept builds on the selective photocaging of a single tyrosine in a nanobody (a single‐domain antibody fragment). Tyrosine is a frequently occurring residue in central positions of the paratope region. o‐Nitrobenzyl‐protected tyrosine variants were incorporated into four nanobodies, including examples directed against EGFR and HER2, and photodeprotection restores the native sequence. An anti‐GFP photobody exhibited an at least 10 000‐fold impaired binding affinity before photodeprotection compared with the parent nanobody. A bispecific nanobody–photobody fusion protein was generated to trigger protein heterodimerization by light. Photoactivatable antibodies are expected to become versatile protein reagents and to enable novel approaches in diagnostic and therapeutic applications.     

Progress and challenge in polymer crazing and fatigue

In this review on polymer crazing and fatigue three aspects have been treated more explicitely: the molecular rearrangements preceding and provoking craze initiation, the competition between disentanglement and chain scission during lateral craze growth, and the distinct fatigue failure mechanism occurring in cyclically loaded PET and PA fibers. An overview on other aspects is given including references to work in progress.     

Entry distribution, fission barrier, and formation mechanism of 254102No

The entry distribution in angular momentum and excitation energy for the formation of 254No has been measured after the 208Pb(48Ca,2n) reaction at 215 and 219 MeV. This nucleus is populated up to spin 22Planck's over 2pi and excitation energy greater, similar6 MeV above the yrast line, with the half-maximum points of the energy distributions at approximately 5 MeV for spins between 12Planck's over 2pi and 22Planck's over 2pi. This suggests that the fission barrier is greater, similar5 MeV and that the shell-correction energy persists to high spin.     

Search for delayed fission products from superheavy nuclei in the reaction136Xe+208Pb

The production of superheavy elements in binary reactions of the type²⁰⁸Pb (¹³⁶Xe, X) Y was investigated atE _c.m.=470 MeV. The experiment was designed to search for delayed fission products from elements withZ between 108 and 116 and fission lifetimes ofΤ?10^?12 s. No fission events were observed the upper limit for the formation cross section being 1.2 Μbarn.     

Radiative lifetimes in Zn(II), Cd(II) and Hg(II) measured by fast-beam zero-field level-crossing technique

The zero-field level-crossing technique has been used to determine radiative lifetimes of excited states in singly ionized zinc, cadmium, and mercury. The excited levels in Zn(II), Cd(II) and Hg(II) were populated by collisions between fast ions and helium gas. Particular attention has been paid to the systematic errors occurring in radiative lifetimes by this technique. The results are compared with the lifetimes obtained by beam-foil, phase-shift, delayed-coincidence techniques and theoretical calculations.     

Temporal integration of loudness in listeners with hearing losses of primarily cochlear origin.

To investigate how hearing loss of primarily cochlear origin affects the loudness of brief tones, loudness matches between 5- and 200-ms tones were obtained as a function of level for 15 listeners with cochlear impairments and for seven age-matched controls. Three frequencies, usually 0.5, 1, and 4 kHz, were tested in each listener using a two-interval, two--alternative forced--choice (2I, 2AFC) paradigm with a roving-level, up-down adaptive procedure. Results for the normal listeners generally were consistent with published data [e.g., Florentine et al., J. Acoust Soc. Am. 99, 1633-1644 (1996)]. The amount of temporal integration--defined as the level difference between equally loud short and long tones--varied nonmonotonically with level and was largest at moderate levels. No consistent effect of frequency was apparent. The impaired listeners varied widely, but most showed a clear effect of level on the amount of temporal integration. Overall, their results appear consistent with expectations based on knowledge of the general properties of their loudness-growth functions and the equal-loudness-ratio hypothesis, which states that the loudness ratio between equal-SPL long and brief tones is the same at all SPLs. The impaired listeners' amounts of temporal integration at high SPLs often were larger than normal, although it was reduced near threshold. When evaluated at equal SLs, the amount of temporal integration well above threshold usually was in the low end of the normal range. Two listeners with abrupt high-frequency hearing losses (slopes > 50 dB/octave) showed larger-than-normal maximal amounts of temporal integration (40 to 50 dB). This finding is consistent with the shallow loudness functions predicted by our excitation-pattern model for impaired listeners [Florentine et al., in Modeling Sensorineural Hearing Loss, edited by W. Jesteadt (Erlbaum, Mahwah, NJ, 1997), pp. 187-198]. Loudness functions derived from impaired listeners' temporal-integration functions indicate that restoration of loudness in listeners with cochlear hearing loss usually will require the same gain whether the sound is short or long.