This page describes two whole-residue hydrophobicity scales determined in our laboratory using measurements of the partitioning of host-guest peptides into bilayer interfaces and n-octanol. The necessity for including peptide-bond contributions to the free energy of partitioning is discussed. The whole-residue octanol scale appears to be a good measure of the partitioning of hydrophobic alpha-helices into the bilayer interior. An example of hydropathy plots of a membrane protein using the interfacial and octanol scales is shown. Our Membrane Protein Explorer (MPEx), a java applet, uses these scales for hydropathy analysis of membrane proteins.

 

Whole-Residue Hydrophobicity Scales

figure 1

Figure 1: For backbone contribution Ac−W−Lm, m = 1 to 6. For side-chain contributions Ac−WL−X−LL.


figure 2

Figure 2

Hydrophobicity scales, composed of experimentally determined transfer free energies for each amino acid, are essential for understanding the energetics of protein-bilayer interactions. Two scales are needed, one for the transfer of unfolded chains from water to bilayer interface and one for the transfer of folded chains into the hydrocarbon interior. The most important feature of practical scales is the inclusion of contributions of the peptide bonds for the obvious reason that whole residues, not just sidechains, partition into membranes. That is, the scales must be whole-residue scales. We have determined such scales for POPC bilayer interfaces (1) and for n-octanol (2) using two families of peptides: host-guest pentapeptides of the form AcWL-X-LL, for determining sidechain hydrophobicities, and the homologous series AcWLm(m = 1..6), for determining peptide bond hydrophobicities (Figure 1). These two scales allow one to compare bilayer interface partitioning with bulk-phase partitioning (1, 3, 4). Used together, these whole-residues scales together appear to be of value in hydropathy plot analyses (4; see below).  The whole-residue transfer free energies determined using the two peptide families are summarized in Figure 2 and Table 1.

 
Table 1. Whole-Residue Hydrophobicity Scales showing the free energies of transfer ΔG (kcal/mol) from water to POPC interface (wif) and to n-octanol (woct).
Amino Acid Interface Scale
ΔGwif (kcal/mol) 		Octanol Scale
ΔGwoct (kcal/mol) 		Octanol − Interface
Scale
Ala 0.17 ± 0.06 0.50 ± 0.12 0.33 ± 0.12
Arg+ 0.81 ± 0.11 1.81 ± 0.13 1.00 ± 0.17
Asn 0.42 ± 0.06 0.85 ± 0.12 0.43 ± 0.13
Asp− 1.23 ± 0.07 3.64 ± 0.17 2.41 ± 0.18
Asp0 −0.07 ± 0.11 0.43 ± 0.13 0.50 ± 0.17
Cys −0.24 ± 0.06 −0.02 ± 0.13 0.22 ± 0.14
Gln 0.58 ± 0.08 0.77 ± 0.12 0.19 ± 0.14
Glu− 2.02 ± 0.11 3.63 ± 0.18 1.61 ± 0.21
Glu0 -0.01 ± 0.15 0.11 ± 0.12 0.12 ± 0.19
Gly 0.01 ± 0.05 1.15 ± 0.11 1.14 ± 0.12
His+ 0.96 ± 0.12 2.33 ± 0.11 1.37 ± 0.16
His0 0.17 ± 0.06 0.11 ± 0.11 −0.06 ± 0.13
Ile −0.31 ± 0.06 −1.12 ± 0.11 −0.81 ± 0.13
Leu −0.56 ± 0.04 −1.25 ± 0.11 −0.69 ±0.12
Lys+ 0.99 ± 0.11 2.80 ± 0.11 1.81 ± 0.16
Met −0.23 ± 0.06 −0.67 ± 0.11 −0.44 ±0.13
Phe −1.13 ± 0.05 −1.71 ± 0.11 −0.58 ± 0.12
Pro 0.45 ± 0.12 0.14 ± 0.11 −0.31 ± 0.16
Ser 0.13 ± 0.08 0.46 ± 0.11 0.33 ± 0.14
Thr 0.14 ± 0.06 0.25 ± 0.11 0.11 ± 0.13
Trp −1.85 ± 0.06 −2.09 ± 0.11 −0.24 ± 0.13
Tyr −0.94 ± 0.06 −0.71 ± 0.11 0.23 ± 0.13
Val 0.07 ± 0.05 −0.46 ± 0.11 −0.53 ± 0.12
 

The Importance of Including the Peptide Bond in Hydropathy Plots

figure 3

Figure 3

The inclusion of the free energy contributions (ΔGHbond) of H-bonded peptide bonds in hydropathy plots of membrane proteins (MPs) is important because ΔGHbond determines the decision level for TM helix selection, as shown here in Figure 3 for the transfer of a glycophorin TM alpha-helix. The total sidechain contribution to the free energy of transfer (−36 kcal/mol) was calculated using the octanol scale. Three estimates for the free energy of transfer of the H-bonded peptide bonds of the backbone are shown along with the assumed value of ΔGHbond in each case. As illustrated, estimates for ΔGHbond as part of an alpha-helix range from +0.6 to +2 kcal mol−1 (see Membrane Protein Folding and Stability). The contribution of the −CH2−CONH−glycyl unit to the partitioning of whole residues into n-octanol is +1.15 kcal mol−1, a value that falls squarely in the middle of the expected range for ΔGHbond. The whole-residue octanol scale may therefore be useful for identifying TM segments in hydropathy plots of MPs, as illustrated below.

 

Whole-Residue Hydropathy Plots: An Example

figure 4

Figure 4

The idea that the octanol scale is useful for identifying TM segments is supported by the whole-residue octanol-scale hydropathy plot for the L-subunit of the photosynthetic reaction center of Rhodobacter sphaeroides shown in Figure 4 (dark yellow solid line). Because TM segments should generally prefer the bilayer rather than water, one would also expect the whole-residue interfacial scale to identify TM segments. This conclusion is supported by Figure 4 (red dotted line). In addition, however, one would expect true TM segments to have more favorable free energies on the octanol scale than on the interfacial scale. Figure 4 (black solid line) shows that this expectation is also met, at least for the L subunit: The hydropathy plot constructed using ΔGwoct − ΔGwif shows favorable peaks on the absolute scale that correspond to the known TM helices. Significantly, the maxima have more restricted sequence-ranges than seen with either of the scales alone and thus identify with less ambiguity the positions of the TM helices. These results make perfect sense: TM helices will choose to be associated with the membrane rather than the water and will prefer a TM location rather than a surface one. Were this not true, MPs would not be stably buried in the membrane. To do hydropathy plots using our whole-residue scales, visit Membrane Protein Explorer (MPEx).

 

References (linked to PubMed)

  1. Wimley WC & White SH (1996). Nature Struct. Biol. 3:842-848.
  2. Wimley WC, Creamer TP & White SH (1996). Biochemistry 35:5109-5124.
  3. White SH. & Wimley WC (1998). Biochim. Biophys. Acta 1376:339-352.
  4. White SH & Wimley WC (1999). Annu. Rev. Biophys. Biomol. Struc. 28:319-365.