Electrostatic Interactions and Binding Orientation of HIV-1 Matrix Studied by Neutron Reflectivity

AbstractThe N-terminal matrix (MA) domain of the HIV-1 Gag protein is responsible for binding to the plasma membrane of host cells during viral assembly. The putative membrane-binding interface of MA was previously mapped by means of mutagenesis and analysis of its trimeric crystal structure. However, the orientation of MA on membranes has not been directly determined by experimental measurements. We present neutron reflectivity measurements that resolve the one-dimensional scattering length density profile of MA bound to a biomimetic of the native viral membrane. A molecular refinement procedure was developed using atomic structures of MA to determine the orientation of the protein on the membrane. The orientation defines a lipid-binding interface consistent with previous mutagenesis results. The MA protein maintains this orientation without the presence of a myristate group, driven only by electrostatic interactions. Furthermore, MA is found to penetrate the membrane headgroup region peripherally such that only the side chains of specific Lys and Arg residues interact with the surface. The results suggest that electrostatic interactions are sufficient to favorably orient MA on viral membrane mimics. The spatial determination of the membrane-bound protein demonstrates the ability of neutron reflectivity to discern orientation and penetration under physiologically relevant conditions

The Take Control Course : conceptual rationale for the development of a transdiagnostic group for common mental health problems

Background: Increasingly, research supports the utility of a transdiagnostic understanding of psychopathology. However, there is no consensus regarding the theoretical approach that best explains this. Transdiagnostic interventions can offer service delivery advantages; this is explored in the current review, focusing on group modalities and primary care settings. Objective: This review seeks to explore whether a Perceptual Control Theory (PCT) explanation of psychopathology across disorders is a valid one. Further, this review illustrates the process of developing a novel transdiagnostic intervention (Take Control Course; TCC) from a PCT theory of functioning. Method: Narrative review. Results and Conclusions: Considerable evidence supports key tenets of PCT. Further, PCT offers a novel perspective regarding the mechanisms by which a number of familiar techniques, such as exposure and awareness, are effective. However, additional research is required to directly test the relative contribution of some PCT mechanisms predicted to underlie psychopathology. Directions for future research are considered

Diagnostic utility of N-terminal TMPP labels for unambiguous identification of clipped sites in therapeutic proteins

Abstract Protein therapeutics are susceptible to clipping via enzymatic and nonenzymatic mechanisms that create neo-N-termini. Typically, neo-N-termini are identified by chemical derivatization of the N-terminal amine with (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) followed by proteolysis and mass spectrometric analysis. Detection of the TMPP-labeled peptide is achieved by mapping the peptide sequence to the product ion spectrum derived from collisional activation. The site-specific localization of the TMPP tag enables unambiguous determination of the true N-terminus or neo-N-termini. In addition to backbone product ions, TMPP reporter ions at m/z 573, formed via collision-induced dissociation, can be diagnostic for the presence of a processed N-termini. However, reporter ions generated by collision-induced dissociation may be uninformative because of their low abundance. We demonstrate a novel high-throughput LC–MS method for the facile generation of the TMPP reporter ion at m/z 533 and, in some instances m/z 590, upon electron transfer dissociation. We further demonstrate the diagnostic utility of TMPP labeled peptides derived from a total cell lysate shows high degree of specificity towards selective N-terminal labeling over labeling of lysine and tyrosine and highly-diagnostic Receiver Operating Characteristic’s (ROC) of TMPP reporter ions of m/z 533 and m/z 590. The abundant generation of these reporters enables subsequent MS/MS by intensity and m/z-dependent triggering of complementary ion activation modes such as collision-induced dissociation, high-energy collision dissociation, or ultraviolet photo dissociation for subsequent peptide sequencing