Alpha-Helix Amphilicity

Alpha-Helix Amphilicity: Relationship Between Hydrophobic Moment, Partitioning, and Helicity

Amphipathic helical peptides bind strongly to membranes. But what is the role of amphiphilicity in interfacial partitioning, and how can it be quantitated? In order to answer these questions, we synthesized two series of peptides: In the first we changed the content of hydrophobic amino acids, and in the second we kept it constant while changing the hydrophobic moment, μH. We started with Ac-(AAQAA)₃-GW-NH₂, μH = 1.7 (A₁₂Q₃-1.7) and gradually increased hydrophobicity by Ala-to-Leu substitutions and produced amphipatic and non-amphipatic amino acid distributions. Only the model peptide, A₈L₄Q₃-4.7, with a high μH of 4.7 bound significantly to POPC membranes. Changing the μH from 2 to 4.7 appears to improve the free energy of transfer by ~5 kcal mol^-1. Therefore, amphilicity is indeed important. The second series of peptides was studied using CD and fluorescence spectroscopy to establish a relationship between μH, partitioning and helicity. Our results show that (1) the helical content in water and in the membrane increases with μH and (2) the total free energy of binding to the membrane decreases with μH. The free energy per residue of helix formation on the membrane is -0.3 kcal mol^-1 per residue and it is the same for all peptides. Previously, we showed the absence of complete additivity of hydrophobic and electrostatic interactions using a series of cationic peptides [Ladokhin and White, (2001) JMB, 309, 543-552]. Because the peptides in the current study bear no charge, the free energy of partitioning into both neutral and anionic vesicles can be compared directly, without interference from Coulombic interactions. We found no difference in the binding to either type of membranes, suggesting that there is no influence of charge on ΔG_μH.

Set of Model Peptides
Name	Sequence	^*μH	^,*ΔG (kcal/mol)
^* Values calculated using the program MPEx.
^** Calculated from Wimley-White hydrophobicity scale [Wimley and White, (199 6) Nature Struct. Biol. 3, 842-848] and corresponds to ΔG_A-to-C in the thermodynamic cycle.
A₈L₄Q₃ -0.55	Ac-LQALAAQALQAAALAGW-NH₂	0.55	-3.44
A₈L₄Q₃ -2.00	Ac-LAQAAALQLLAAQAAGW-NH₂	2.00	-3.44
A₈L₄Q₃ -2.86	Ac-AQLAALAALQAAQLAGW-NH₂	2.86	-3.44
A₈L₄Q₃ -4.72	Ac-AAAQAAAQLLQALLAGW-NH₂	4.72	-3.44
A₈L₄Q₃ -5.51	Ac-QLAQALAAALAALAQGW-NH₂	5.51	-3.44
A₈L₄Q₃ -5.54	Ac-QALQALAAALAALAQGW-NH₂	5.54	-3.44

Helical Wheel and Hydrophobic Moment

μH = 2 (non-amphipathic)	μH = 4.72 (amphipathic)

Helical Content

CD spectra in water of the peptides with hydrophobic moment of 0.55, 2.86 and 5.51, measured at 298K.

Helicity in Water Increases with the Hydrophobic Moment
Name	^,*[Θ]_222nm	%Helix	^,**[Θ]_222nm	%Helix
^* Units of [Θ] are deg cm²dmol^-1.
^** Values of molar ellipticities in water.
^*** Values of molar ellipticities in the membrane.
A₈L₄Q₃ -0.55	-4400	9%	-12000	30%
A₈L₄Q₃ -2.00	-6500	15%	-13000	35%
A₈L₄Q₃ -2.86	-8200	20%	-16000	45%
A₈L₄Q₃ -4.72	-10700	28%	-24000	69%
A₈L₄Q₃ -5.51	-13500	37%	-24700	71%
A₈L₄Q₃ -5.54	-14000	38%	-25000	72%

Some of our peptides have Leu pair interactions (i, i+3) and (i, i+4) which stabilize the helices [Luo and Baldwin, (2002) Biophys. Chem., 96, 103]. Applying MPEx to their peptides, we observed the same effect: Increasing the μH increases the helicity:

Increased Hydophobic Moment Increases Helicity
Name	Side-Chain Interaction	%Helix	μH
Ac-KAAAAKAALAKLAAAKGY-NH₂	(i, i+3>	26%	0.57
Ac-KAAAAKAALAKALAAKGY-NH₂	(i, i+4)	33%	1.33
Ac-ELAALKAKLAALKAKAGY-NH₂	2(i, i+3) + (i, i+4)	43%	1.48
Ac-ELAALKAKLAALKAKLGY-NH₂	2(i, i+3) + 2(i, i+4)	51%	1.62

Partitioning Measured By CD and Flourescence


CD spectra of A₈l₄Q₃ - titrated with POPC LUV. Increasing the concentration of lipid increases the helical content, measured as molar ellipticity at 222nm.		Binding curve obtained from the titration of A₈L₄Q₃ with POPC LUV. The binding curve gives the maximum helical content in the membrane and the binding constant. The free energy of Gibbs is calculated as: ΔG = -RTlnK.


Flourescence spectra of A₈L₄Q₃ -5.54 with the maximum position at 350nm (corresponding to Trp in water). Adding 3mM POPC LUV, the position shifts to 335 nm, indicating binding to the membrane.		Binding curve of the titration with POPC LUV of A₈L₄Q₃ -5.54. The binding constant and the free energy are obtained, and they are consistent with the values obtained by CD.

Thermodynamic Analysis

ΔG_H = ΔG_H(H₂O) - m[alcohol] [Hirota et al., (1997) Protein Sci. 6:416-421):

Hydrophobic Moment Thermodynamics Graph Legend

Conclusions

Helical content in solution and in the membrane interface increases with the hydrophobic moment.
Free energy of partitioning into the membrane, decreases with the hydrophobic moment: ΔG_f = ΔG_A-to-D + K[μH] = -(4.5 ± 0.2) - (0.48 ± 0.06)×[μH] For melittin: μH = 5.18 => ΔG_f = -7.0 ± 0.5 kcal mol^-1 (in agreement with previous results).
The total free energy of helix formation in water, decreases with the hydrophobic moment but the per residue value remains constant for all the peptides (~+0.1 kcal mol^-1 res^-1).
There is no difference in binding into neutral and charged membranes, suggesting there is no influence of charge on ΔG_μH.
From analysis of the thermodynamic cycle, we have found that the free energy of helix formation on the membrane interface is -0.33 kcal mol^-1 per residue and it is the same for all the peptides.

Acknowledgements

This work was supported by NIH grants GM 46823 and GM 069783.