Date of Award
Doctor of Philosophy
Solid-state NMR is among the most important analytical techniques to provide atomic-level structural and dynamic information of chemical and biological systems. Due to the insoluble and non-crystalline nature of most membrane peptides and proteins, SSNMR is particularly powerful to investigate their conformations, dynamics, domain assembly, oliogmerization, and the characteristic structural properties in lipid bilayers including insertion orientation and depth, residue-lipid interaction, and membrane per-turbation. Our research is to collect structural and dynamic information and correlate it with biological functions to elucidate the structure-bioactivity relation. In my PhD pro-jects, we have successfully applied various SSNMR techniques to study many interesting membrane peptides including the cell-penetrating peptide (CPP), antimicrobial peptides (AMP), antimicrobial oligomer (AMO), gating helix of K+ channel (KvAP) and trans-membrane 1H channel of influenza M2 protein (M2TM). We also developed a novel paramagnetic-ion-membrane bound paramagnetic relaxation enhancement (PRE) method to provide quantitative long-range distance constrain (~20 y) in membrane-active bio-systems and applied the method to obtain high-resolution residue-specific insertion depth of two membrane peptides, penetratin and M2TM.
One main category of my research topics is the cationic membrane peptide. On the one hand, phospholipid membranes have highly hydrophobic interiors that cannot accommodate charged species, while on the other hand, cationic peptides need to insert or translocate across the membrane to conduct biological functions. So, we are motivated to uncover the structural basis of the membrane insertion and translocation. With this motivation, we have studied two kinds of cationic bio-macromolecules, including CPP and AMP. We have experimentally proved that all these Arg-rich peptides generally have strong guanidinium-phosphate interaction with the phospholipids. This charge-charge interaction causes headgroup reorientation and allows the peptide to insert. For CPPs, the guanidinium-phosphate ion pair helps to stabilize the unstructured peptide in the membrane-water interface. The observed peptide-water interaction further minimizes the peptide polarity and makes it more membrane-soluble. We find that two representative CPPs, penetratin and TAT, have highly dynamic and plastic conformations, proposed to facilitate the movement within the membrane. In the penetratin study, the one-side Mn2+-bound PRE method has been developed and applied to study the pep-tide-concentration dependent insertion depth and symmetry in the outer and inner leaflets of the POPC/POPG bilayer. Another important kind of cationic membrane peptides is AMP. Taking PG-1 and its charge reduced mutant IB484 as model AMPs, we have stud-ied the antimicrobial mechanism, and for the first time, provided high-resolution struc-tural information to elucidate the bacterial Gram-selectivity. We find that the interaction manifests the manner of peptide insertion in terms of orientation and depth, which in turn determined the antimicrobial ability in gram positive and negative bacterial membranes. The antimicrobial mechanism of a guanidinium-rich AMO, PMX30016, has also been investigated. The finding of drug-concentration dependant lipid 31P CSA change and the fast uniaxial motion in the interfacial membrane region suggest a subtle and combined antimcicrobial mechanism of membrane potential perturbation and in-plane disruption.
Another category of my research topics is the transmembrane ion-conductive channel study, including the gating mechanism of a K+ channel (KvAP) and the blocking mechanism of the M2TM 1H channel by the metal ion inhibitor (Cu2+). We have deter-mined the topology of an isolated gating helix (S4) of KvAP and compared the orientation with that of an intact K+ channel, Kv1.2-Kv2.1 paddle chimera. The identical tilted and rotational angles of the S4 helix in the isolated form and intact protein, and the observed interaction suggest the channel gating might be manifested by the pep-tide-lipid interaction rather than the interaction among different helical domains. Finally, we applied PRE techniques to study the Cu2+-inhibited M2TM channel and obtained high resolution Cu2+ binding structure and long-range distance constraints for the binding structure refinement.
Su, Yongchao, "Structure and dynamics of membrane peptides from solid-state NMR" (2011). Graduate Theses and Dissertations. 10197.