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The membrane proximal region (MPR, residues 649–683) and transmembrane domain (TMD, residues 684–705) of the gp41 subunit of HIV-1’s envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662–683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649–705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM).
Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41.
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Methods: The Eye to Eye mentorship program assessed involved mentors and mentees who completed 12 in-person art sessions out of the normal 20 in-person sessions. The first main assessment was the BLD (Breger Learning Difference) Feedback Survey addressing one’s experience in the Eye to Eye program and which were completed at the end of the mentorship program and filled out by mentors, mentees, and mentees parents (one parent for each mentee). A total of 12 mentors, 6 mentees, and 6 mentee parents were included in the feedback survey final analysis. The second main assessments were the pre and post Behavior Assessment System for Child, Third Edition (BASC-3) provided to mentors, mentees, and mentees parents (one parent for each mentee). A total of 10 mentors, 5 mentees, and 5 mentee parents were included in pre and post BASC-3 final analysis. Fall 2019 (pre) and Spring 2020 (post) optional interviews involved 5 mentors and 3 mentees who showed interest and were comfortable participating with additional release forms.
Results: The program was generally positively rated in the feedback survey by mentors, mentees, and mentee parents. The highest responses for mentors, mentees, and mentee parents all incorporated average ratings of 4.0 or higher (out of 5.0) for perceived understanding of socio-emotional skills after Eye to Eye, experience in Eye to Eye, how having a mentor or mentee made them feel, and perceived change in self-awareness. All three groups reported fairly high ratings of improved self-awareness of 4.0/5.0 or above. No negative ratings were provided by any participants and the lowest response was no change. The BASC-3 evaluation found statistically significant improvement in mentors’ anxiety and atypicality and mentees’ sense of inadequacy.
Discussion: The Eye to Eye program was popular and well-rated despite only involving 12 in- person one-hour art sessions. The mentors, mentees, and mentee parents felt positive about the Eye to Eye program when answering the feedback survey. Some suggestions are made on how to improve this program to better enhance someone with learning differences future ability to succeed. Future research is needed to assess the true impact due to the COVID-19 epidemic and other limitations.
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