Despite their apparent simplicity, coiled coil (Coiled Coil) modules are highly specialized and are important in understanding tertiary structure and its formation. The general type of structure of the most commonly observed coiled coil form, the parallel dimerization state, remains to be fully described. Nonetheless, its structure has shown the strict rule of requiring certain specific types of amino acids at certain positions. The source of this experiment is "A Guide to Modern Protein Engineering Experiments" [German] K.M. Arndt, K.M. Miller, eds.
Operation method
Design and optimization techniques for coiled spiral structures Move This section discusses several different aspects involved in the design of coiled-coil helix specificity. Our goal is to select amino acids at the core and at the edge positions to obtain the desired oligomerization state (see 3. 2.1), specificity (see 3. 2. 2 ), and helix orientation (see 3.2.3 ). Here, we also relate different design options for specific stability. Subsection 4 (see 3.2.4) deals with overall stability, focusing on the external sites (b, c and f ). In this chapter we have divided all the contents into subsections and discussed them one by one. However, it is due to the nature of the discussed interacting factors leading to the formation of coiled-coil helices that the discussions in this section cross each other in several places. Thus, the subsections dealing with the different roles played by the same residue position can be considered as interconnected fragments. For more product details, please visit Aladdin Scientific
3. 2. 1 Oligomeric states
To function properly, any protein must fold into a suitable three-dimensional structure. Similarly, coiled coil structures must take on the correct oligomeric (four-level) structure. In the next subsection, the major factors in achieving the correct structure are discussed.
3.2.1.1 Core residues
In Figure 3.1, we call the a and d residues the core residues because they form a hydrophobic band around each helix. The nonpolar nature of the a and d repeating residues favors oligomerization along one side of each helix. This is analogous to the hydrophobic core that curls up into during the folding of globular proteins and represents a major contribution to the overall stability of the coiled-coil helices. The result is that the core residues play a major role in defining the oligomerization state.
The hydrophobic side chains on positions a and d are buried in neighboring helices in a "pestle-and-pestle fit mortar" (knob- into - hole) fashion and were first described by Crick in 1953 [ 15 ]. In this model, one side chain of an a-helix (pestle) is inserted into a space (mortar) surrounded by four side chains on the opposite a-helix, and vice versa. The stacking geometry is defined by the projection at the bottom of the helix of the angle formed by the Cα-Cβ bond and the Cα-Ca vector at the bottom of the mortar. For a parallel dimer, the pestle at position a (the seven-membered repeat position i) fits into the mortar of the other helix made up of the residues in the position arranged clockwise (if viewed against the cavity). Thus, the pestle at position di is in a mortar surrounded by a 'i, d' i, e ' i, and a' i+1 (Figure 3.1).
A comprehensive and complete analysis of the different core mutations in the GCN4 ( the yeast homologue of the Jun transcription factor, sometimes referred to as GCN4-p1, see Note 1) coiled-coil helix revealed different stacking geometries for the different oligomerization states (Table 3.1; Ref. [ 16]).Comparison of the side-chain stacking in the X-ray structure of the GCN4-p1 dimer with that of a designed tetrameric GCN4 mutant demonstrates that that the local geometries of the a- and d-layers are flipped. Parallel pestle-adapted white stacking is found in the 3-layer of the dimer and the d-layer of the tetramer. In contrast, perpendicular pestle-adapted mortar stacking is found in layer d of the dimer and layer a of the tetramer. In the parallel trimer, a third type of pestle-adapted mortar stacking is found in the a and d positions [17], where the Cα-Cβ bonds of each pestle in both layers are at an approximate 60° angle to the Cα-Cβ bonds at the bottom of the corresponding mortar. This arrangement is referred to as "sharp" pestle-adapted mortar stacking. 
These different geometrical relationships explain the unique amino acid preferences of certain oligomeric states. Below are the results of several experiments that tested the effect of different amino acids on stability and oligomerization specificity.
( 1 ) Specific hydrophobic residues are critical in forcing the coiled-coil helix to adopt which oligomerization state.Harbmy et al. systematically changed all a and b residues of GCN4 ( except a1Met ) to Leu, Val, or lie ( see Note 2 ; ref. [16] ). As shown in Table 3.1, this allows the coiled-coil helix to adopt different oligomerization states. the GCN4- IL (IL refers to lie in the a-position and Leu in the d-position), II, and LI mutants are dimeric, trimeric, and tetrameric, respectively, and are concentration-independent over the entire range of concentrations studied (oligomerization states). the VI, VL, LV, and LL mutants oligomerize in a variety of ways. Each combination of L, V, and I gives a unique stacking preference and thus a unique geometric profile.
a. Residues with β-branching (Val and lie) are suited for parallel mortar-and-pestle stacking (a layer of dimers and d layer of tetramers), while residues with γ-branching (Leu) are suited for perpendicular (d layer of dimers and a layer of tetramers) geometries. In contrast, the β-branched amino acids insert into vertical positions and require thermodynamically unfavorable rotational conformations [18].
b. It is likely that Lie and Val, although having similar stereochemistry, are not equivalent in the creation of oligomeric states. Val in the a-site is less capable of leading to dimerization than lie. In contrast to Val, which leads to dimer-trimer mixtures, lie gives higher dimer-specific interactions.
c. In contrast to dimeric and tetrameric structures, the stacking within the trimer, at the two hydrophobic sites, can accommodate β-branching residues with optimal rotational conformational states.
( 2 ) Included in Woolfson and Alber's study were studies of the roles of the core residues and the use of these roles as a measure of the tendency of the seven-membered repeating residues to dimerize and trimerize [19]. The properties of dimers and trimers were analyzed and used to identify new sequences. Buried Leu, lie, Asn, Lys, and Gin are key in its prediction algorithm, called COILER. overall, 21 different proteins known to form dimers and trimers were used, equivalent to the 721 heptameric repeats in the database.
a. Of the seven amino acids considered initially (Ala, Phe, lie, Leu, Met, Val ), only lie and Leu at the a-position and Leu at the d-position were observed in a statistically significant way. b. Of the seven amino acids considered initially (Ala, Phe, lie, Leu, Met, Val ), only lie at the a-position was observed in a statistically significant way. The dimer preferences are lie-enriched at the a position and Leu-enriched at the d position, whereas there is almost no lie at the d position in the dimer. it is clear that these choices of amino acids at these positions result in the best stacking geometries for the formation of the dimer [see 3.2.1.1 in (1 ) and Table 3.1].
b. Val is more evenly distributed than lie at the core of the coiled-coil helical sequence. At positions a and d of dimers and tetramers, Val occurs even less frequently than expected from a random distribution. These results are consistent with the observation that it is difficult to distinguish between dimeric and trimeric states of the coiled-coil helix for Val at positions a and b [Section 3.2.1.1 (1) and Table 3.1].
c. The stacking of a- and d-positions in the trimer is similar, and these positions are less specific for amino acids, resulting in a more uniform distribution of hydrophobic amino acids [16, 19].
( 3 ) In studying the positional effects of alanines at the core of a designed antiparallel coiled-coil helix (see Note 3 ), Monera et al. found that dimers are formed when alanine residues are in the proper position (i.e., on the same ring) [ 20 ]. If the alanine residues are not synchronized, a tetramer is formed. The most likely explanation for this is that the pores formed by synchronized alanines in tetramers are highly unstable and thus tend to form dimers, whereas the Leu-Ala repeats distribute the pores over a larger area, thus reducing losses in hydrophobic burial and van der Waals interactions. This demonstrates that oligomerization specificity is caused by core residue stacking.
( 4 ) Cartilage Oligomeric Matrix Protein (COMP ), which belongs to the platelet-responsive family of proteins, contains a 5-chain, very stable, parallel α-helix coiled-coil structure.The 46 amino acid-long coiled-coil helix region (see Note 4 ) contains intercyclic (i.e., helix-to-helix) cysteine disulfide bonds [21]. The pentameric interface exhibits pestle-adapted mortar stacking. a-, d-, e-, and g-residues form a number of pestles that are stacked into a mortar surrounded by the a'-g', d'-e', c'-d', and a'-b' sidechains of the neighboring subunits. Only the residue at position f is fully exposed, whereas the residues at the other six positions are clearly buried.Both the GCN4-pLI mutant [ see (1 ) in 3.2.1.1 ] and the pentameric COMP structure contain a large axial pore. The tunnel in the tetramer has a size of 1.0 to 1.3 A [16] and excludes water molecules (radius 1.4 A) as a result. In contrast, there are several small molecules along the pore in pentamers, which is consistent with the larger radius of their tunnels (2 to 6 A).
( 5 ) Experiments changing the d residue to non-natural amino acids that are more hydrophobic in nature (trifluoroleucine and hexafluoroleucine) show an increase in stability (see Note 5; references [ 22] and [ 23]).
( 6 ) Hydrophobic burial at the a/d interface was studied with mono-, di-, and trimethylated analogs of diaminopropionic acid (dap), which showed an increase in hydrophobicity [22]. The addition of a methyl group to the 16th position of a monomer (aspartic acid was used in the 16th position of the peptide analog) resulted in a more stable heterodimerization of the subsequently obtained GCN4-p1 ( see Note 6 ), probably due to the increase in van der Waals interactions of the folded state and the reduction of solvation losses of the fold. However, the addition of 3 methyl groups decreased the stability, probably due to increased stereochemical site resistance. Curiously, the addition of 2 methyl groups to the synthesized dap induced zwitterionization (the binding of identical proteins or peptides is called zwitterionization or homodimerization). This demonstrates that even small changes in scale and hydrophobicity can alter stability and folding tendency.
In short, in a coiled-coil helix of dimerization, the best choice of amino acid for the core position may be Leu at the d-position, lie at the a-position ( β-branch (or Val). If Val is used, Asn (as commonly seen in natural coiled-coil helices) should be placed in the central a position accordingly to increase the specificity of the interaction (see 3.2.1.2 ). The best design for trimers is to use lie at all core positions, while tetramers are preferred to use Leu at the a-position and lie at the d-position. Deviations from this β/y side-chain branching (residue) arrangement result in unfavorable rotational isomerism, reduce the desired structural stability, and may produce a mixture of oligomeric and antiparallel binding states of the coiled-coil helix, which results in a decrease in specificity.
3.2.1.2 Polar core residues
In contrast to the overall hydrophobicity of the core residues, about 20% of the core residues are charged [ 25 ]. These charged residues usually serve to enable the structure to form the correct oligomerization state, presumably by ensuring that the helices are arranged in the structure in the desired manner, otherwise the structure would exist as dimerization, trimerization and/or tetramerization, as well as as parallel and antiparallel mixtures. However, the usual gain in specificity is always accompanied by a decrease in stability. The roles of these residues in specificity and stability are listed below.
( 1 ) In a statistical analysis of dimers and trimers [ see 3.2.1.1( 2 ) ], Woolfson and Alber observed:
a. Lys and Asn are favored in the a-position in dimers, while they are largely absent in trimers. Asn is three times more likely to be found in the a-site of dimers than in trimers.
b. The a-site of the trimer is enriched in Gln, while Ser and Thr are enriched in the a-site or d-site.
c. Buried lysine is often found in the a-site along with glutamate in the lateral e-site and g-site. This can be found, for example, in the Jun/Fos heterodimer [26] as well as in the X-ray structure of the GCN4 Asn6Lys mutant [see 3.2.1.2 ( 5 ); Ref. [27]].
( 2 ) Buried Asn pairs (when in the right position) may give specificity to the dimer through hydrogen bonds generated between the two Asn side chains of the two helices, a phenomenon indeed observed in X-ray [6] and NMR [28] studies. Other conformations that do not satisfy the hydrogen bonding site energy of the Asn side chains are therefore energetically unfavorable.
( 3 ) If Asn in the a-site of the GCN4 -p1 core (see Note 2) is replaced by Val, the stability of the coiled-coil helix increases substantially at the expense of dimerization specificity. Because Val with the lie a-site renders specificity absent, Harbury et al. reported obtaining a mixture of dimers and trimers [ see 3.2.1.1 ( 1 ) and Table 3.1; Ref. [16] ]; meanwhile, Potekhin et al. reported trimer generation [29] .
( 4 ) In another example, the core Asn pair of a parallel heterodimeric peptide (see Note 1 ) was mutated to a Leu pair (yielding Acid- pLL and Base-pLL polypeptides), yielding a mixture of parallel and antiparallel tetramers [ 30 ].
( 5 ) In the case of GCN4 (see Note 2 ), the Alber group altered the Asn pair in the core a-site to Gln and Lys to see if this also yielded oligomerization specificity. lys produces a dimer similar to wild-type, whereas Gln produces a mixture of dimers and trimers [27]. They reasoned that the structural uniqueness constrained by the polar moiety is not only caused by polar burial but also depends on the correct interaction of the side chain with the environment. These context-dependent effects are much more difficult to predict than just the residue frequency of a given seven-membered repeat position.
( 6 ) When selecting coiled-coil helices for heterodimerization with a protein fragment's complementary reagent (see 3. 2. 5. 3 ) dihydrofolate reductase, Arndt et al. found that coiled-coil helices at a core that was Val-Leu were preferred over cores made up of Asn-Val or Val-Val-combined Asn pairs (see Note 7; Ref. [31]). This is consistent with many natural coiled-coil helices.
( 7 ) In another study, the a-position or d-position of dimeric GCN4-pVL [see 3.2.1.1 ( 1 )] was mutated to the polar residues Asn, Gln, Ser, or Thr, respectively (see Note 8; Ref. [25]). Only the Asn residue pair in the a-position and the Thr residue pair in the d-position lead to the correct oligomerization state. It is possible that the solvation energy lost in burying these residues in the core is compensated by the interaction energy between them, thus also ensuring the correct helical arrangement.
( 8 ) Differences in stacking environments give hydrophobic residues different tendencies, even at the a and d positions. In two thorough studies applying model peptides with Val in the a-position and Leu in the d-position, the central a- and d-positions were systematically altered for each amino acid to test their roles in stability and oligomerization (see Note 9; references [ 32 ] and [ 33 ]). These alterations, the first thorough quantitative assessment of the effect of hydrophobic core side-chain substitutions on the stability of double-stranded coiled-coil helices, made it possible to establish thermodynamic relative stability scales for the 19 natural amino acids at the a-position and d-position. Table 3.2 lists those amino acids which, if placed in either the a- or d-position, result in a well-defined oligomeric state [32]. a-position Leu-, Tyr-, Gin-, and His- substitution analogs are invariably three-chained, whereas Asn-, Lyt-, Om-, Arg-, and Trp- substitution analogs invariably form two-chained monomers. At central d-site substitutions, lie and Val (β-branching residues) result in triple-stranded oligomers [see 3.2.1.1(1)], while Tyr, Arg, Lys, Orn, Glu, and Asp produce double-stranded states. 
( 9 ) Ji et al. mutated gp41, a 6-helix envelope protein from simian immunodeficiency virus, which, together with gp120, is responsible for fusion of the virus with CD4+ cells [ 34 ]. Structurally, it is a trimeric protein composed of antiparallel heterodimers. In this study, each of the four buried polar residues (two Gln and two Thr residues) responsible for the core hydrogen bonds and salt bridges was individually mutated to lie. of these, three form more stable 6-helix bundles and the other one forms an insoluble aggregate (see Note 10). These results demonstrate the ability of these residues to control the balance between structural stability and specificity. This is important because this protein undergoes structural changes prior to fusion and must have the correct balance of stability between the two structures to make such changes possible. These polar core residues assist in regulating this conformational stability and, therefore, in the membrane fusion itself.
In general, the Asn residue pair in the a-site of the core is clearly dominant in the dimer, especially if the core deviates from the optimal Ile-Leu a-d residue arrangement, since this combination appears to lead to parallel dimerization without the need for the Asn pair within the core. The a-site Gln pair trimer of the core may be a good choice, although some other factors may be required to ensure the specificity of the trimer exclusion.
3.2.1.3 Edge residues
The e- and g-positions (edges) of the heptad repeat flank the a- and d-residues at the interface of the coiled-coil helix (Figure 3.1). The burial of these positions is highly dependent on the oligomerization state. As a result, the selection of amino acids at the e- and g-positions may be influenced by the oligomerization state. Table 3.3 shows the percentage of buried area, indicating the fraction of side chain accessible surface area in the presence of the helix alone that becomes buried in the oligomeric state [16] . 
( 1 ) The e- and g-positions of the trimer are enriched in hydrophobic residues (lie, Leu, Val, Phe, Tyr, and Trp) and are almost devoid of specific hydrophilic residues (Glu, Gln, Ser, and Arg; Ref [ 19 ] ) compared to the corresponding positions in the dimer. These patterns are consistent with the expansion of the hydrophobic interface in trimers relative to dimers. The increase in the percentage of hydrophobic residues increases the width of the narrow hydrophobic surface, and the likelihood of the high oligomerization states increases. There is more nonpolar burial in the high oligomerization state than in the two-helix coiled coil. This can be seen in Table 3.3. The percentage of hydrophobic burial at the e-site and g-site has increased by about 40%. This is consistent with the decrease in the gi to e 'i+1 residue pairs charged on the opposite side in the trimer (12% ) relative to the dimer (23% ) [19].
( 2 ) Fairman et al. mutated the C-terminal homotetrameric coiled-coil structural domain of the lac deterrent protein to produce heterotetrameric coiled-coil helices [ 35 ]. Peptides with all Lys or all Glu in the b and c positions around the e and g positions are weakly bound. However, when mixed together, they form highly stable tetramers (see Note 11). This demonstrates that, at least for the tetramer, the b and c residues also play an important role in the stability of the coiled-coil helix. This role is similar to the g/e' ionic interactions present in dimeric coiled helices, but the widened hydrophobic interface of the tetramer extends to these residues, and the ionic interactions start at the core and recede outward to the b and c residues. These ion-pair interactions between Glu and Lys, by varying pH and salt levels, were shown to be responsible for the increase in stability. In addition, these charges are directly opposite each other in zwitterionic oligomerization, and it is likely that this unfavorable charge repulsion drives the formation of heterodimers in potential zwitterionic interactors [ 9, 35].
3.2.2 Pairing specificity
This section discusses the importance of pairing specificity and some of the ways that coiled-coil helices can be used to ensure that no other energetically favorable structure is acceptable. Strikingly, in contrast to sequence and structural similarity, coiled-coil helices favor interactions with functional partners. This section analyzes the factors that mediate this high specificity.
3.2.2.1 Core residues
As described in subsection 3.2.1.1, the pattern of distribution of hydrophobic residues (mainly Leu, lie, and Val ) is the main driving force for interhelical binding. However, because this pattern is so common, how can a helix simultaneously use its core to resist the formation of different structures? The answer is a complex picture containing subtle variations, such as the insertion of unstabilized, non-hydrophobic core residues, which choose to fight against other kinds of structures.
( 1 ) Sharma et al. designed a peptide (anti-APCp1) that was designed to be used to bind the coiled-coil helix sequence of the intestinal cancer-associated tumor-suppressor protein from adenomatous polyps of the colon (APC ) (see Note 12; Ref. [36]). In doing so, they used the core alteration as well as the binding g/e' interaction, rather than the Asn pairing, to confirm that specificity could be attributed to this interaction. They reasoned that given the dominant role of core residues in the binding process, and the fact that core mutations have a large effect on both stability and specificity, it was surprising to find a low requirement for core residues in terms of dominant specificity. To deal with this, they designed 1 peptide to bind to the first 55 amino acids of APC (APC55 ) and mutated this anti-APCp1 based on the covariation pattern of type I and type II heterodimers of keratins at positions a and d to produce the more common a-a' and d-d' pairings. They made three mutations (A41 I in the a-layer, A2M and M44A in the d-layer) to change the wild-type Ala-Ala and Met-Met interactions to the more common Ala-lie, Ala-Met and Met-Ala interactions, respectively. Two further mutations (T6G in the a-layer and N30H in the d-layer) destabilize the zwitterion with corresponding Gly-Gly and His-His pairs. The additional e-g' pair optimizes the ionic interactions while targeting the anti-anti-APCp1 zwitterionization, and the resulting dimer is stable and specific.
( 2 ) Schnarr and Kennan formed heterotrimeric proteins by stereochemical coordination of core hydrophobic residues [37] . In their study, a variety of unnatural amino acids with different side chain lengths were used to initiate specific heterotrimer formation. The researchers replaced the GCN4 core a-position residue with Ala or cyclohexylalanine (see Note 13), resulting in a stereochemically mismatched core layer in the trimer to 3 Ala or 3 cyclohexylalanine, producing steric avoidance or repulsion. However, 2 Ala and 1 cyclohexylalanine produced 1 heterotrimer with a good stereochemical fit. Unnatural side chains can produce coiled-coil helices in this manner. The extra volume of the cyclohexylalanine compensates for the Ala core layer to provide stereochemistry, while the larger side chain only destabilizes the molecule.
3.2.2.2 Polar core residues
The role of polar core residues in directing helix-specific oligomerization was discussed in 3.2.1.2. As mentioned in 3.2.1.1, heterotypic core contacts in coiled-coil helix pairing allow for heterospecificity. More specificity can be obtained by the interaction of the polar core with the outer edge residues.
( 1 ) In natural dimeric coiled-coil helices, Lys at the a position is the most common buried polar residue after Asn [19].Lys at the A position can form intra-helical electrostatic interactions with residues at the e position in the first heptad repeats [27], as well as inter-helical g'-a polar interactions with polar residues at the g' position in the first heptad repeat of the opposite helix in parallel dimers [26].
( 2 ) Campbell and Lumb placed two a-position Lys in the Base-pLL of the peptide zipper (PV) to achieve interhelical polar interactions in the Acid-pLL of the peptide zipper provided by these two Lys with the Glu residues in the e' and g' positions (see Note 14; Ref. [38]). As expected, the results are skewed toward the dimeric state, most likely because of the larger solvation losses that occur in the highly oligomeric state. Also, such interactions, compared to Asn-Asn (a-a') interactions, are less detrimental to stability, mainly because the latter have larger solvation losses to pay. However, both parallel and antiparallel interactions are possible, mainly because the a-g' interaction in the parallel orientation is energetically similar to the interaction in the antiparallel orientation.
( 3 ) Harbury's group used a computational approach [ see 3.2.5.4 (4) ] to achieve specificity by taking into account not only desired structures, but also different undesired structures [39]. The a-, d-, e-, and g-position residues of the central seven-membered repeat of GCN4 were altered (see Note 15 ); all non-proline residues as well as homozygous and heterozygous sequences were computationally selected and experimentally verified. Subsequent to the bulk and charge complementarity of the g/e' residue pair, Glu in the d-position was found to pair preferentially with Arg in the e' position.
3.2.2.3 Marginal residues
Pairing specificity is affected by the electrostatic nature of the e- and g-position residues (in a parallel dimerization helix, the g-position of a seven-membered repeat with the e'-position of the next seven-membered repeat of the other helix is noted as i→i'+5 ). These residues are often Glu and Lys, respectively.Such complementary pairs of residues interact to add to and consolidate, respectively, the specificity and stability originally provided by core hydrophobic interactions. The charge pattern at the contact edges outside the coiled-coil helix guides whether the protein forms a zwitterionic or heterodimeric protein, and whether the orientation within the coiled-coil helix is parallel or antiparallel. However, the PV assumption (see Section 1; Ref. [ 8 ] ) is oversimplified; residues that have little or no role in complex stability serve their purpose by directing helices to avoid, those that would jeopardize the specificity of the molecule, the same or undesired interactions (negative design). On the other hand, certain coiled-coil helices do not appear to have survived evolutionary pressures because they are already specific and are not required to be more stable than their present state.
( 1 ) As expected, the replacement of the favorable g/e' position Gin-Gin pairing with the repulsive Glu-Gk pairing has been shown to destabilize the coiled-coil helix conformation [ 40].
( 2 ) Hodges and colleagues estimated the contribution of the salt bridge of the g/e' pair to the stability of the coiled-coil helix to be 0.37 kcal/mol (see Note 16; Ref. [41]).
( 3 ) Careful charging of the e and g sites allows for the generation of heterodimers and additionally ensures that zwitterions do not tend to be generated, as encompassed by the PV assumption [ 9, 42, 43] .
( 4 ) The four most common amino acids found in the e- and g-positions are Glu, Gln, Arg, and Lys; these residues contain long hydrophobic side chains that interact with the a and b core residues and terminate in a charged (Glu, Arg, or Lys) or polar (Gin) group. By mutating e, then g, and finally Ala on the last two seven-membered repeat pairs, Krylov et al. succeeded in establishing the coupling energies of these intercontacting residues for the chicken yolk proteogen-binding protein (see Note 17). This double mutant thermodynamic cycle was used to establish a thermodynamic scale for the propensity of the outer edge residues to contact (Table 3.4). The Glu-Arg attraction proved to be slightly more stable than the Glu-Lys attraction at 150 mmol KCl and pH 7.4, reasonably explained by the fact that the side chain of Arg is longer and interacts better with glutamate, and that the individual methyl groups shield the core from the solvent more effectively. This has the consequence of increasing the role of the charged end group, producing a larger contribution in the weaker aqueous environment. As expected, high salt like low pH - like, weakens these interactions. At high salt or low pH, polar interactions are weakened and hydrophobic interactions are enhanced. the Glu-Arg, Glu-Lys and Gin-Gin interactions are the most stabilizing (independent of orientation). Glu-Glu, Arg-Arg, Arg-Lys, Lys-Arg, and Lys-Lys, which are all identically charged, are rather unpopular. 
( 5 ) A peptide library was designed by Arndt et al. The design of this library is based on Jim-Fos heterodimerization, with residues b, c, and f derived from the respective wild-type proteins, Val and Leu at positions a and d (with the exception of the insertion of a3Asn in the core, which directs the desired helical orientation and oligomerization), and residues e and g altered with trinucleotides to give equimolar mixtures of Arg, Lys, Gin, and Glu (see Note 7). and reference [8]). Surprisingly, the best selected successor, the Winzip-A2B1 heterodimer (see Note 18), lacks fully complementary charged residues at the g/e' position, although the selection process was thorough. More precisely, two of the six g/e' pairs are predicted to be repulsive, suggesting that the resulting sequence deviates from the simple charge complementation rule (PV assumption). It can be assumed that the overall electrostatic potential (both intramolecular and intermolecular interactions) plays a major role, and that interactions with core residues, such as those like favorable stacking or steric impulses, can also modulate these g/e' interactions (see Refs. [ 8] and [ 31] and references cited therein). Such observations are consistent with naturally occurring coiled-coil helices, natural structures that often have complex interaction patterns. These coiled-coil helices must fulfill criteria such as biological stability and ver