Conformational Ensembles of Calmodulin Revealed by Nonperturbing Site-Specific Vibrational Probe Groups.

Seven native residues on the regulatory protein calmodulin, including three key methionine residues, were replaced (one by one) by the vibrational probe amino acid cyanylated cysteine, which has a unique CN stretching vibration that reports on its local environment. Almost no perturbation was caused by this probe at any of the seven sites, as reported by CD spectra of calcium-bound and apo calmodulin and binding thermodynamics for the formation of a complex between calmodulin and a canonical target peptide from skeletal muscle myosin light chain kinase measured by isothermal titration. The surprising lack of perturbation suggests that this probe group could be applied directly in many protein-protein binding interfaces. The infrared absorption bands for the probe groups reported many dramatic changes in the probes' local environments as CaM went from apo- to calcium-saturated to target peptide-bound conditions, including large frequency shifts and a variety of line shapes from narrow (interpreted as a rigid and invariant local environment) to symmetric to broad and asymmetric (likely from multiple coexisting and dynamically exchanging structures). The fast intrinsic time scale of infrared spectroscopy means that the line shapes report directly on site-specific details of calmodulin's variable structural distribution. Though quantitative interpretation of the probe line shapes depends on a direct connection between simulated ensembles and experimental data that does not yet exist, formation of such a connection to data such as that reported here would provide a new way to evaluate conformational ensembles from data that directly contains the structural distribution. The calmodulin probe sites developed here will also be useful in evaluating the binding mode of calmodulin with many uncharacterized regulatory targets.

Messages About Abstinence, Delaying Sexual Debut and Sexual Decision-Making in Conversations Between Mothers and Young Adolescents.

STUDY OBJECTIVE: Little is known about the information shared during family discussions about sexuality. From a public health perspective, abstinence is one of the most important sexuality topics parents can talk about with adolescents. We sought to characterize the messages mothers communicate to young adolescents regarding abstinence. DESIGN: Content analysis of dyadic discussions that occurred between June 2011-December 2012 between mothers and their 10- to 14-year-old adolescent sons and daughters. Discussions were audio-recorded, transcribed, and a grounded theory approach to content analysis performed. SETTING: Urban city in Western Pennsylvania. PARTICIPANTS: Twenty-one dyads; 15 mother-daughter dyads and 6 mother-son dyads. INTERVENTIONS: None. MAIN OUTCOME MEASURES: None. RESULTS: Four key themes emerged reflecting the high priority mothers placed on abstinence, delaying their adolescent's sexual debut, and nurturing sexual decision-making skills. Theme 1 focused on ensuring that adolescents understand what abstinence means. In defining abstinence, only 1 mother explained what sex is. The 3 remaining themes emphasized sexual decision-making and emphasized when it is acceptable to stop being abstinent (theme 2), why abstinence is important (theme 3), and mothers' desire to engage in ongoing discussions, particularly when an adolescent was considering becoming sexually active (theme 4). Messages did not vary according to mothers' age or according to adolescent age, gender, or race. CONCLUSION: Mothers convey complex information about abstinence and sexual decision-making to young, non-sexually active adolescents. Message tailoring on the basis of the adolescents' age or sex was not observed.