The C-terminal amphipathic helix of the influenza A M2 protein plays a critical cholesterol dependent role in viral budding. To provide atomic-level detail on the impact cholesterol has on the conformation of M2 protein, we spin-labeled sites right before and within the C-terminal amphipathic helix of the M2 protein. We studied the spin-labeled M2 proteins in membranes both with and without cholesterol. We used a multipronged site-directed spin-label electron paramagnetic resonance (SDSL-EPR) approach and collected data on line shapes, relaxation rates, accessibility of sites to the membrane, and distances between symmetry related sites within the tetrameric protein. We demonstrate that the C-terminal amphipathic helix of M2 populates at least two conformations in POPC/POPG 4:1 bilayers. Furthermore, we show that the conformational state that becomes more populated in the presence of cholesterol is less dynamic, less membrane buried, and more tightly packed than the other state. Cholesterol dependent changes in M2 could be attributed to the changes cholesterol induces in bilayer properties and/or direct binding of cholesterol to the protein. We propose a model consistent with all our experimental data that suggests that the predominant conformation we observe in the presence of cholesterol is relevant for the understanding of viral budding.

Sarcomeres, the basic units of striated muscle cells, are built of a highly ordered filament system which includes myosin, actin, and titin. Titin is the largest known protein with~34800 amino acids which gives muscles some of its elastic properties [1]. Titin is also involved in several signaling pathways via interactions with a number of proteins. Its interaction with MARPs proteins is very important as these interactions are involved in stretch signal in muscle [2]. The MARP proteins, specifically MARP2 is present in sarcomere of skeletal muscles and binds to N2A region of titin [3]. The N2A region consists of four Ig domains I-80 to I-83 and intervening/unique sequence 'IS' between Ig domains I-80 and I-81. The structure of N2A region is currently unknown but critical for stress sensing. MARP2 binds to the N2A-IS region in a relaxed state and is dissociated when a muscle is overstretched [2]. This binding is also disrupted in case of the murine mdm deletion [4] which deletes part of the I-83 Ig domain and part of the PEVK region. We are characterizing the N2A-IS region using a combination of CD, FRET and solution state NMR as well as in silico methods. The in silico analysis showed that N2A-IS region consists of a-helices and some of the regions are disorder. These results were also confirmed by CD experiments, which confirmed the a-helical nature of this region. Ongoing solution state NMR studies on U-15N and U-13C labelled N2A-IS constructs will provide high-resolution structural information on this region.