Date of Award
Doctor of Philosophy
Solid-state NMR is often applied to study many aspects of membrane proteins in biologically relevant model membrane systems including the structure, dynamics, and oligomerization. Solid-state NMR is commonly used for membrane proteins in lipid bilayers because these systems are difficult to study by other structure specific biophysical methods such as x-ray crystallography and solution NMR. In this thesis, we apply solid-state NMR to explore the properties of arginine rich membrane peptides in unaligned lipid vesicles and aligned lipid bilayers. Multiple solid-state NMR techniques have also been applied to study water-peptide, lipid-peptide and water-lipid interactions in these systems.
The antimicrobial peptide tachyplesin I (TP-I) is an amphipathic peptide constrained to a β-hairpin structure by two disulfide bonds. Here it is studied extensively along with its linear mutants that had the cysteines replaced by other amino acids. Wild type TP-I was found to be a linear β-hairpin that is inserted in the interfacial region of the lipid bilayer with an orientation parallel to the bilayer surface. TP-I was found to severely disrupt bacterial mimicking POPE/POPG bilayers by micellizing the bilayers while not disrupting mammalian mimetic bilayers. The linear mutants that had alanine (TPA4) and tyrosine (TPY4) replacing the cysteines caused non-selective disruption without micellization of the bilayer. The mechanism of action of these peptides was explored by various magic angle spinning (MAS) experiments and it was found that the antimicrobial activity of these peptides did not correlate with structure or insertion depth, but did correlate with the dynamics of the peptide. The active peptides, TP-I and TPF4, are more mobile in the bilayer than the inactive peptide TPA4 is, suggesting that large-amplitude motions are critical to the antimicrobial activity of the tachyplesins.
The isolated S4 helix from the voltage gated voltage sensor membrane protein KvAP is another arginine rich peptide studied. It is a mostly hydrophobic α-helical sequence with 4 arginine residues evenly spaced along the helix. The peptide was determined to have a tilt angle of 40 y 5° and a rotation angle of 280 y 20° in lipid bilayers. Based on lipid 31P to peptide 13C distance measurements the peptide was found to cause membrane thinning of ~9 Å. This membrane thinning likely occurs to allow the charged sidechains of arginine to access the lipid-water interface and reduce contact with the hydrophobic bilayer core.
Lipid-water interactions in vesicles were investigated by a 2D 31P-1H correlation experiment. Chemical exchange was found to be necessary for magnetization transfer from water to lipid, as observed by water-lipid cross peaks only in lipids with labile protons. The presence of charged peptides in the bilayer was found to shorten the water 1H T1. This was attributed to slow peptide motion, intermolecular hydrogen bonding, and chemical exchange with the labile protons on the peptide.
We developed a new method to observed heteronuclear spectra of uniaxial rotating lipids and peptides in lipid vesicles by combining moderate MAS and very low power 1H decoupling. This technique opens the door for studying membrane peptides on solution NMR spectrometers that are not equipped with expensive high-power 1H amplifiers.
Timothy Franklin Doherty
Doherty, Timothy Franklin, "Investigations of cationic peptides in lipid membranes by solid-state NMR" (2009). Graduate Theses and Dissertations. 10675.