# On the Helix-Coil transition in grafted chains

## Abstract

The helix-coil transition is modified by grafting to a surface. This modification is studied for short peptides capable of forming $`\alpha `$-helices. Three factors are involved: (i) the grafting can induced change of the boundary free energy of the helical domain (ii) the van der Waals attraction between the helices and (iii) the crowding induced stretching of the coils. As a result the helix-coil transition acquires “all or nothing” characteristics. In addition the transition temperature is elevated and the transition itself sharpens as the grafting density increases.

During the past two decades the physics of polymer brushes formed by terminally anchored chains were studied extensively. Most of the research effort dealt with brushes of flexible, synthetic polymers, devoid of internal degrees of freedom. In contrast, this letter concerns brushes formed by biopolymers capable of undergoing a cooperative helix-coil transition. It is motivated by two experimental observations. First, the promotion of the adhesion and spreading of cells by brushes of collagen model peptides. Second, membrane fusion induced by model fusogenic peptides grafted to vesicles. In both cases, the function of the short peptide chains was correlated with a helical state. Furthermore, the helical state was favored by the grafting. With this in mind we present a highly simplified theoretical model for the helix-coil transition in brushes of short, laterally immobile peptides. In particular, we discuss the transition temperature $`T_t`$ and the width of the transition $`\mathrm{\Delta }T`$ as a function of grafting density, $`\mathrm{\Sigma }^1`$. We focus on the simplest situation, of short neutral homopeptides forming a single stranded $`\alpha `$-helix. As we shall see, the grafting of the chains can lead to qualitative modifications of the helix-coil transition: (i) in marked distinction to the case of a free peptide, the helix-coil transition in an isolated grafted peptide can acquire “all or nothing” characteristics; (ii) $`T_t`$ is elevated with the grafting density while $`\mathrm{\Delta }T`$ decreases; (iii) eventually, for high grafting density the transition may take place as a first-order phase transition. These distinctive features arise because of three factors: The lower configurational entropy of the monomer at the grafting site favors helix formation even in isolated chains. In a brush, the helical state is also promoted by the crowding induced stretching and by the attractive van der Waals interaction between the helices.

In a $`\alpha `$-helix  the $`i`$th monomer forms $`H`$-bonds with the $`(i3)`$th and the $`(i+3)`$th monomers. Overall, a helical domain consisting of $`n`$ monomers contains $`n2`$ $`H`$-bonds. It is convenient to consider the chain as a sequence of bonds. Choosing the coil state as a reference state, each helical bond is associated with an excess free energy $`\mathrm{\Delta }f`$ reflecting the formation of $`H`$-bonds, the accompanying change in solvation and the loss of configurational entropy. $`\mathrm{\Delta }f`$ is a function of the temperature, $`T`$. $`\mathrm{\Delta }f`$ vanishes at the transition temperature $`T_{}`$, while $`\mathrm{\Delta }f<0`$ when $`T<`$ $`T_{}`$ and $`\mathrm{\Delta }f>0`$ for $`T>T_{}`$. The terminal bonds, at the boundary of the helical domains, are associated with an additional free energy penalty $`\mathrm{\Delta }f_t`$. This arises because the terminal bonds lose their configurational entropy but do not contribute $`H`$-bonds. $`\mathrm{\Delta }f_t`$ plays the role of an interfacial free energy associated with the helix-coil boundary. It is customary to formulate the theory of the helix-coil transition in terms of the Bragg-Zimm parameters $`s=\mathrm{exp}(\mathrm{\Delta }f/kT)`$ and $`\sigma =\mathrm{exp}(2\mathrm{\Delta }f_t/kT)`$. $`\sigma `$ is independent of $`T`$ and is typically of order of $`10^310^4`$, depending on the identity of the amino acid residues forming the peptide. On the other hand, $`s`$ is a function of $`T`$ and in the vicinity of $`T_{}`$ it varies as $`s1(T_{}T)/T_{}`$. In an infinite chain, the plot of the fraction of helical bonds, $`\theta `$ vs. $`s`$ is sigmoid and the width of the transition is $`\mathrm{\Delta }TT_{}\sigma ^{1/2}`$. Since the chain is one dimensional object, a first order phase transition is impossible. As a result the chain consists of an alternating sequence of helical and coil regions. The minimal size of a domain comprising a helical and a coil regions, as obtained at $`T_{}`$, is $`\sigma ^{1/2}`$. When the polymerization degree, $`N`$ is much larger than $`\sigma ^{1/2}`$ the chain may be considered as infinite. It consists of a large number of domains and the width of the transition, $`\sigma ^{1/2}`$, is independent of $`N`$. On the other hand, when $`N\sigma ^{1/2}`$ the chain incorporates typically only one helical region and the width of the transition is of order $`1/N`$. The statistical physics of such short oligopeptides are well described by the “one sequence approximation” where the chain is assumed to contain a single helical region . The customary formulation of this approximation utilizes the appropriate partition function. For the purposes of our discussion it is convenient to utilize the corresponding free energy. The chemical potential of a chain supporting a helical domain consisting of $`n`$ bonds is $`\mu _0(n)=n\mathrm{\Delta }f+2\mathrm{\Delta }f_tkT\mathrm{ln}(Nn1)`$ where the last term is the entropy associated with the placement of the helical segment along the chain. Altogether there are $`N2`$ bonds and the first monomer of the helical segment of length $`n`$ can be placed in any of the $`(N2)n+1`$ sites. For $`n=0`$ we have $`\mu _0(0)=0`$. The fraction of chains with a given $`n`$, $`p_n`$, is determined by minimizing $`\mathrm{\Omega }=\mathrm{\Omega }_0+M_np_n[\mu _0(n)+kT\mathrm{ln}p_n]`$ subject to the constraint $`_np_n=1`$. This leads to $`\mu =\mu _0(n)+kT\mathrm{ln}p_n=const^{}`$ since all species coexist in equilibrium. For our discussion it is sufficient to consider the dominant term in $`\mathrm{\Omega }`$, as specified by $`\mu _0(n)/n=0`$. When $`\mathrm{\Delta }f0`$ the minimal $`\mu _0(n)`$ is $`\mu _0(0)=0`$ and the majority of chains contain no helical region. On the other hand, for $`\mathrm{\Delta }f<0`$ the probability distribution peaks at $`n_{}=N1kT/\mathrm{\Delta }f`$. Accordingly, $`n_{}`$ attains its maximum value, $`n_{}=N2`$, when $`\mathrm{\Delta }f=kT`$. The discussion as presented above applies to free chains. Two features are of special importance for future reference: (i) $`\mathrm{\Delta }f_t`$ is independent of the position of the helical region and, as a result (ii) all placements of the helical segments are equally probable. Because of the associated entropy $`n_{}`$ is lower than $`N2`$ for $`0>\mathrm{\Delta }f>kT`$.

The situation described above is modified significantly when the chain is grafted, terminally anchored, to a surface. We first discuss the case of a single chain grafted to a flat surface. The grafting gives rise to two effects. First, the overall number of configurations of the chain is reduced because of the presence of an impenetrable wall. As a result, the overall configurational entropy is reduced by $`\mathrm{\Delta }S\mathrm{ln}N`$. This is expected to lead to a small changes in $`s`$ and $`\sigma `$ which we will ignore since they do not give rise to qualitative effects. Second, the two boundaries of the helical region are no longer equivalent because of the presence of the surface. The surface effect is expected to decay with the length of the coil region separating the helical region from the wall. The maximal effect is attained when the helical region is initiated at the wall. The penalty of the “free” boundary remains $`\mathrm{\Delta }f_t.`$ However, the terminal penalty at the wall, $`\mathrm{\Delta }f_w,`$ can be different. The difference can arise from two sources. First a modification of the torsional potential for the terminal monomer and the consequent reduction of its configurational entropy. Second, change in the electrostatic interactions experienced by the terminal monomer at the grafting site. As a result, the Bragg-Zimm parameters are modified and for a helical sequence at the wall $`\sigma _g=\mathrm{exp}[(\mathrm{\Delta }f_w+\mathrm{\Delta }f_t)/kT].`$ In the following we consider the physically plausible case of $`\mathrm{\Delta }f_w<\mathrm{\Delta }f_t`$. For simplicity we assume that $`\mathrm{\Delta }f_w`$ is attained only when the helical sequence begins at the grafting site. In this situation a helical sequence of length $`n`$ may assume two states: (i) When the terminal monomer is not at the wall, the helical sequence can be freely placed at the available remaining sites. The “free” sequence is associated with $`\mu _{f0}=n\mathrm{\Delta }f+2\mathrm{\Delta }f_tkT\mathrm{ln}(Nn2)`$. (ii) A “bound” state when the terminal monomer is at the grafting site and $`\mu _{b0}=n\mathrm{\Delta }f+\mathrm{\Delta }f_t+\mathrm{\Delta }f_w`$. The two states are equally stable when $`\mu _{f0}=\mu _{b0}`$. This condition is satisfied for $`n_{}`$ given by $`n_{}=N2\mathrm{exp}[(\mathrm{\Delta }f_t\mathrm{\Delta }f_w)/kT]`$. For $`n>n_{}`$ the bound state is favored. Accordingly, the bound state is always dominant when $`n_{}=1`$ or $`\mathrm{ln}(N3)(\mathrm{\Delta }f_t\mathrm{\Delta }f_w)/kT`$. Furthermore, among the bound states, the fully helical state is always the most stable i.e., $`\mu _{b0}(N)<\mu _{b0}(n)`$ whenever $`\mathrm{\Delta }f<0`$. For this choice of $`N`$ the coil state remains the most probable state, $`n_{}=0`$ when $`\mathrm{\Delta }f>0`$ while for any $`\mathrm{\Delta }f<0`$ the most probable state is fully helical, $`n_{}=N2`$. Thus, the grafting of short chains modifies the nature of the helix-coil transition that acquires “all or nothing” characteristics. In the remainder of this letter we focus on brushes of such chains.

Our earlier considerations concerned a single grafted chain. With them in mind we model a grafted layer consisting of many chains as a mixture of chains in a coil state and chains in a fully helical state. In this case, additional contributions come into play. One is the van der Waals attraction between the rods. Another important contribution is the crowding induced extension of the coils in the brush regime. To quantify this picture it is necessary to introduce further assumptions concerning the orientation imposed on the helices by the grafting. For simplicity we will consider the case of grafting sites enforcing perpendicular orientation of the helices with respect to the surface. We further limit the discussion to the case of rigid junctions that is, the helices cannot bend at the grafting sites. Thus far, our considerations were directed at chains with $`N\sigma ^{1/2}10^2`$. At this point it is necessary to limit our discussion further to chains with $`NN_{}(a/a_h)^2`$ where $`a`$ is the effective monomer size in the coil state while $`a_h=1.5`$Å is the projection of a residue on the axis of the helix. While $`a`$ depends on the identity of the residue, $`a/a_h>1`$ and typically $`N_{}50`$. This choice is necessary because the length of a fully helical chain, $`L`$, is smaller than the radius of the corresponding random coil, $`R_0N^{1/2}a`$ when $`NN_{}`$ while $`L>R_0`$ if $`N>N_{}`$. Thus, in one case the helices are submerged in the brush formed by the coils while in the other the helices can be partially exposed (Figure 1). This choice of the $`N`$ range is dictated by the experimental systems motivating this work . With the system fully specified we are in a position to write down the free energy per chain, $`F`$. For simplicity we use a modification of the Alexander model , assuming that the coils are uniformly stretched and that concentration profile of the monomers of the coils is step-like. We consider a planar grafted layer such that the area per chain is $`\mathrm{\Sigma }`$ and the fraction of chains in a helical state is $`\theta `$. $`F`$ consists of four terms, $`F=F_0+\theta F_{vdW}+(1\theta )F_{cc}+\theta F_{hc}`$. $`F_0`$ reflects the contribution of the non interacting chains $`F_0\theta (N\mathrm{\Delta }f+\mathrm{\Delta }f_t+\mathrm{\Delta }f_w)+kT[\theta \mathrm{ln}\theta +(1\theta )\mathrm{ln}(1\theta )]`$ where the last term is the mixing entropy associated with the possible placements of chains in the two possible states. The van der Waals attraction between two oriented helices is modeled as the interaction between two parallel rods of diameter $`d`$. When the distance between two neighboring rods, $`D`$, is small in comparison to $`L`$, their length, $`F_{vdW}L/D^5`$ while in the opposite limit $`F_{vdW}L^2/D^6`$. In the first case, an element of the rod experiences, in effect, an interaction with an infinite neighboring rod. In the second limit, $`F_{vdW}`$ reflects the sum of all pairwise interactions between the elements of the two rods. The van der Waals energy of a rod within the grafted layer, where the minimal distance between two neighboring helices is $`(4\mathrm{\Sigma }/\pi \theta )^{1/2},`$ is

$$F_{vdW}=\frac{ALd^4}{18}\left(\frac{\pi \theta }{4\mathrm{\Sigma }}\right)^{5/2}\mathrm{arctan}\sqrt{\frac{\pi \theta L^2}{4\mathrm{\Sigma }}}$$
(1)   
where $`A`$ is the Hamaker constant. In a “normal” brush, the overlap between the coils gives rise to chain extension along the normal to the surface. The stretching reduces the number of monomer-monomer contacts at the price of an extension penalty. In our situation the picture is somewhat different. First, it is necessary to allow for both coil-coil and coil-helix interactions. Second, only the coils are extendible. Finally, for our choice of $`N`$, the helices are fully immersed in a brush of coils. $`F_{cc}`$ allows for the elastic free energy of the coils as well as for monomer-monomer interactions involving the coils. This contribution has the form of the free energy of a brush formed by coils that is, $`F_{cc}/kTH^2/Na^2+v_{cc}N^2a^3(1\theta )/H\mathrm{\Sigma }`$ where $`H`$ is the thickness of the brush and $`v_{cc}`$ is the virial coefficient for monomer-monomer interactions involving coils. The first term reflects the Gaussian stretching penalty while the second allows for the repulsive monomer-monomer contacts. $`F_{cc}`$ must be supplemented by $`F_{hc}`$ that reflects the interactions between the coils and the helices, $`F_{hc}/kTv_{hc}N^2a^3(1\theta )/H\mathrm{\Sigma }`$. Here $`v_{hc}`$ is the virial coefficient for the binary interactions between helical monomers and coil monomers. For simplicity we consider the case of $`v_{hc}=v_{cc}=v`$. In this case the equilibrium thickness of the brush $`H_{eq}/aN(va^2/\mathrm{\Sigma })^{1/3}`$, as specified by $`F/H=0`$, is independent of $`\theta `$ so long as $`\theta `$ is small enough to ensure overlap between the coils. For such a choice of $`\theta `$ the equilibrium form of the free energy per chain, as obtained by substituting $`H_{eq}`$ into $`F`$, is

$`{\displaystyle \frac{F}{NkT}}`$ $``$ $`{\displaystyle \frac{\theta \mathrm{\Delta }f}{kT}}+{\displaystyle \frac{\theta (\mathrm{\Delta }f_t+\mathrm{\Delta }f_w)}{NkT}}\lambda \theta ^{7/2}\left({\displaystyle \frac{a^2}{\mathrm{\Sigma }}}\right)^{5/2}+(1\theta )\left({\displaystyle \frac{va^2}{\mathrm{\Sigma }}}\right)^{2/3}`$ (2)   
$`+{\displaystyle \frac{1}{N}}\left[\theta \mathrm{ln}\theta +(1\theta )\mathrm{ln}(1\theta )\right]`$   
where $`\lambda \frac{ALd^4}{NkTa^5}`$. $`F_{vdW}`$ in (2) is approximated by the $`\pi \theta L^2/\mathrm{\Sigma }1`$ limit of (1). As a result, $`F_{vdW}`$ is overestimated for $`\theta 1`$. This does not affect our analysis since the contribution of the van der Waals attraction in this regime is negligible. The corresponding spinodal condition, $`^2F/\theta ^2=0`$, leads to $`f(\theta )=\theta ^{5/2}(1\theta )(4/35)(\mathrm{\Sigma }/a^2)^{5/2}(\lambda N)^1`$ revealing a critical point at $`\theta _c=5/7`$ where $`f(\theta )`$ exhibits a maximum. The critical grafting density is thus specified by

$$\mathrm{\Sigma }_c/a^2\lambda ^{2/5}N^{2/5}$$
(3)   
and for $`\mathrm{\Sigma }<\mathrm{\Sigma }_c`$ the helix-coil transition within the layer proceeds as a first-order phase transition. For lower grafting densities, $`\mathrm{\Sigma }>\mathrm{\Sigma }_c,`$ the helix-coil transition is cooperative but no phase transition is involved. In this last regime it is of interest to characterize the dependence of the transition temperature $`T_t`$ and the width of the transition $`\mathrm{\Delta }T`$ on $`\mathrm{\Sigma }`$ and $`N`$. To this end it is helpful to recast the equilibrium condition, $`F/\theta =0`$, in the form

$$\frac{\theta }{1\theta }=K(\theta )s^N\sigma _g\mathrm{exp}\left[\frac{7}{2}\lambda N\left(\frac{\theta a^2}{\mathrm{\Sigma }}\right)^{5/2}+N\left(\frac{va^2}{\mathrm{\Sigma }}\right)^{2/3}\right]$$
(4)   
where $`K(\theta )`$ is the equilibrium constant governing the ratio of helices and coils. A rough idea concerning the transition is obtained by identifying it with the condition $`\theta =1/2`$ i.e., $`K(\theta )=1`$. It is helpful to consider first the case of non interacting mushrooms, $`\mathrm{\Sigma }\mathrm{}`$, when $`K(\theta )=s^N\sigma _g`$. Since $`s`$ in the vicinity of $`T_{}`$ is given by $`s\mathrm{exp}\left[r\left(\frac{T}{T_{}}1\right)\right]`$ where $`r`$ is a phenomenological constant, this condition leads to

$$T_t(\mathrm{})T_{}+\frac{T_{}}{Nr}\mathrm{ln}\sigma _gT_{}.$$
(5)   
Similarly we define the width of the transition as $`\mathrm{\Delta }T=T_{}T_+`$ where $`T_+`$ is the temperature for which $`\theta _+=9/10`$ while $`T_{}`$ is the temperature corresponding to $`\theta _{}=1/10`$. The ratio $`K(\theta _+)/K(\theta _{})=\left(s_+/s_{}\right)^N=81`$ may be rewritten as $`\mathrm{ln}81Nr\mathrm{\Delta }T(\mathrm{})/T_{}`$ or

$$\mathrm{\Delta }T(\mathrm{})\frac{T_{}}{Nr}.$$
(6)   
By using the same argument for a brush with $`\mathrm{\Sigma }R_0^2`$ we obtain

$$T_t=T_t(\mathrm{})+\frac{T_{}}{r}\left[\frac{7}{2}\lambda \left(\frac{a^2}{2\mathrm{\Sigma }}\right)^{5/2}+\left(\frac{va^2}{\mathrm{\Sigma }}\right)^{2/3}\right]$$
(7)   
and

$$\mathrm{\Delta }T=\mathrm{\Delta }T(\mathrm{})\frac{7}{2}\frac{T_{}}{r}\lambda \left(\frac{9a^2}{10\mathrm{\Sigma }}\right)^{5/2}.$$
(8)   
As $`\mathrm{\Sigma }`$ decreases, $`T_t`$ increases while $`\mathrm{\Delta }T`$ decreases. In other words, the stability of the helical state grows and the transition becomes sharper when the grafting density increases. Eventually, at the critical point, $`\mathrm{\Delta }T=0`$ thus signaling the onset of a phase transition. Note that within our model the decrease of $`\mathrm{\Delta }T`$ is due only to $`F_{vdW}`$ while the increase of $`T_t`$ results from contributions from $`F_{vdW}`$ as well as the brush penalty. This is because our choice of $`v_{hc}=v_{cc}`$ leads to a $`\theta `$ independent $`H_{eq}`$. In the general case $`\mathrm{\Delta }T`$ should reflect both contributions.

The analysis presented above focused on the case of short, immobile, grafted chains that form a single-stranded helix. In this system $`T_t`$ increases while $`\mathrm{\Delta }T`$ decreases as the grafting density grows. The effect of grafting on $`\sigma `$, the van der Waals attraction and the crowding induced stretching should however play a role irrespective of the precise specifications of the grafted layer. Additional factors may contribute when chain mobility, multiple stranded helices and longer chains are considered. Lateral chain mobility may give rise to in-plane phase separation driven by the van der Waals attraction. The discussion of multiple stranded helices should allow for the effect of loops on $`\sigma `$. It may also be necessary to allow for loss of translational entropy due to the formation of a multiple helix. The “all or nothing” model described above is clearly limited to brushes of short chains. A discussion of brushes formed by long helix-forming chains should allow for helix-coil coexistence on each of the polymers. While the discussion focused on brushes of peptides, it should be noted that similar situation is encountered in DNA chips undergoing hybridization. Finally, it is of interest to note that our considerations are somewhat similar of the analysis of the coupling between the helix-coil transition and the onset of liquid crystalline order in peptides solutions and in a collapsing chain. From this perspective, the distinguishing features of the brush case are due to the grafting modification of $`\sigma `$ and to the crowding induced chain stretching.

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The authors benefited from instructive discussions with M. Tirrell and D. Leckband.