Kinetics of hydrogen bond breakage in the process of unfolding of ribonuclease A measured by pulsed hydrogen exchange.

A sensitive test for kinetic unfolding intermediates in ribonuclease A (EC 3.1.27.5) is performed under conditions where the enzyme unfolds slowly (10 degrees C, pH 8.0, 4.5 M guanidinium chloride). Exchange of peptide NH protons (2H-1H) is used to monitor structural opening of individual hydrogen bonds during unfolding, and kinetic models are developed for hydrogen exchange during the process of protein unfolding. The analysis indicates that the kinetic process of unfolding can be monitored by EX1 exchange (limited by the rate of opening) for ribonuclease A in these conditions. Of the 49 protons whose unfolding/exchange kinetics was measured, 47 have known hydrogen bond acceptor groups. To test whether exchange during unfolding follows the EX2 (base-catalyzed) or the EX1 (uncatalyzed) mechanism, unfolding/exchange was measured both at pH 8.0 and at pH 9.0. A few faster-exchanging protons were found that undergo exchange by both EX1 and EX2 processes, but the 43 slower-exchanging protons at pH 8 undergo exchange only by the EX1 mechanism, and they have closely similar rates. Thus, it is likely that all 49 protons undergo EX1 exchange at the same rate. The results indicate that a single rate-limiting step in unfolding breaks the entire network of peptide hydrogen bonds and causes the overall unfolding of ribonuclease A. The additional exchange observed for some protons that follows the EX2 mechanism probably results from equilibrium unfolding intermediates and will be discussed elsewhere.

The Tube on YouTube: TV Series, Media Strategies, and User Tactics in a Transmedia Environment

This study analyzes the traffic generated on YouTube around television series. We selected a sample of 314 short YouTube videos about 21 Spanish TV series that premiered in 2013 by Spain’s three most popular mainstream television networks (Telecinco, Antena 3, and La1). These videos, which together received more than 24 million views, were classified according to two key variables: the nature (official or nonofficial) of the YouTube channel on which they were located and the exclusivity of their content (already broadcast on TV or Web exclusive). The analysis allows us to characterize the strategies used by TV networks on YouTube and the activity of fans as well as their efforts in the construction of a transmedia narrative universe around TV series.