Masters Theses

Date of Award

5-1997

Degree Type

Thesis

Degree Name

Master of Science

Major

Physics

Major Professor

Solon Georghiou

Committee Members

Elizabeth Howell, Engin Serpersu, Thomas D. Bradrick

Abstract

Melittin is a naturally occurring protein in honey bee venom. The association of melittin with phospholipid vesicles is well documented. The present work uses its single tryptophan residue as an intrinsic fluorescent probe. Measurements were taken at the transition temperatures of the phospholipids used, 21° C and 37° C for DMPC and DPPC, respectively. In order to study the depth of penetration of melittin into the lipid bilayer, we have used the n-doxyl stearic acids, n-DS, as fluorescence quenchers (n defines the position along the acyl chain of the fatty acid). The greater the value for n the deeper into the bilayer the quencher moeity is located. Stopped-flow fluorometry has been used in the present work to study the quenching on the millisecond time scale before melittin-induced fusion takes place.

The relative quenching efficiencies for the differing depths of the n-DS quenchers are used to infer the accessibility of the tryptophan of melittin to different depths into the bilayer. It is found that the further down the acyl chains one puts the quencher the less efficient it is for both DMPC and DPPC. These findings imply that the tryptophan residue of melittin resides closer to the 5 position along the acyl chain than to either the 7 or 12 position.

Quenching efficiency is found to be dependent upon which lipid is used in conjunction with the n-DS. The n-DS are much more efficient when they are incorporated into DPPC vesicles rather than DMPC vesicles. This is thought to stem from a tighter binding of melittin which may reduce the distance between its tryptophan and the quenchers. The results of data analysis using the parallax method indicate that melittin inserts itself into approximately the first position along the acyl chains in DMPC vesicles. However, the parallax method gives unrealistic results for DPPC, as it places the tryptophan at a distance of 30 A away from the center of the bilayer. It should be added that for this lipid there is a significant contribution of instantaneous quenching of unknown origin. However, regardless of whether one takes into account the instantaneous quenching, a comparison of the quenching efficiencies of 5-DS and 12-DS suggests that the position of the tryptophan is closer to the bilayer surface and hence more external when binding to DPPC.

Interestingly the rate of quenching was also found to be dependent upon the location of the n-DS. It was found that the further down the acyl chain one puts the quencher the faster it quenches. This is attributed to the enhancement of segmental flexibility which the methylene groups closer to the center of the bilayer are known to possess. It should also be noted that the overall rate of quenching was found to be much higher for the n-DS in DPPC than in DMPC, which correlates with published results for the rate of melittin binding.

The relative orientation of the protein once bound to a vesicle is still unknown. However, the present experiments have provided information on the extent of penetration of the protein into the bilayer. The suggested deeper penetration in DMPC tends to favor the parallel-to-the-surface model rather than the other models, where the N-terminus would presumably limit the degree of freedom for the melittin to change its depth of insertion.

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