Helix‐capping interaction in λ cro protein: A free energy simulation analysis

Abstract

The stability mutant Tyr-26-->Asp was studied in the Cro protein from bacteriophage lambda using free energy molecular dynamics simulations. The mutant was calculated to be more stable than the wild type by 3.0 +/- 1.7 kcal/mol/monomer, in reasonable agreement with experiment (1.4 kcal/mol/monomer). Moreover, the aspartic acid in the mutant was found to form a capping interaction with the amino terminus of the third alpha-helix of Cro. The simulations were analyzed to understand better the source of the stability of this helix-capping interaction and to examine the results in light of previous explanations of stabilizing helix caps--namely, a model of local unsatisfied hydrogen bonds at the helix termini and the helix macrodipole model. Analysis of the simulations shows that the stabilizing effect of this charged helical cap is due both to favorable hydrogen bonds with backbone NH groups at the helix terminus and to favorable electrostatic interactions (but not hydrogen bonds) with their carbonyls (effectively the next row of local dipoles in the helix). However, electrostatic interactions are weak or negligible with backbone dipolar groups in the helix further away from the terminus. Moreover, the importance of other local electrostatic interactions with polar side chains near the helix terminus, which are neglected in most treatments of this effect, are shown to be important. Thus, the results support a model that is intermediate between the two previous explanations: both unsatisfied hydrogen bonds at the helix terminus and other, local preoriented dipolar groups stabilize the helix cap. These findings suggest that similar interactions with preoriented dipolar groups may be important for cooperativity in other charge-dipole interactions and may be employed to advantage for molecular design.

Helix capping

“…Thus, capping is independent of helix length (i.e., charge separation in the macrodipole). Tidor (1994) also found that electrostatic contributions from the macrodipole decrease markedly beyond the first/last turn, based, in this case, on simulation of a well-characterized mutation in A Cro (Pakula & Sauer, 1990). By calculating contributions to the free energy from an Ncap mutation (viz., YZ6 + D in the helix that spans residues 26-36), he concluded that the capping effect is due both to specific hydrogen bonding and to electrostatic interactions with spatially proximate groups, some nearby in sequence, some quite distant.…”

The denaturation and degradation of stable enzymes at high temperatures

“…the α-helix, success may be closer. Introduction of a negatively charged residue near the N-terminus of an α-helix has a stabilizing effect due to enhanced electrostatic and hydrogen bonding interactions within the helix [25,33,[38][39][40][41]. Given the fundamental role played by α-helices, this may be a useful stabilizing strategy, although substitution at the ends of the α-helices may require a very good knowledge of the local stereochemistry to allow such a stabilization strategy to be used reliably.…”

“…The hydrophobic effect plays a major role in enzyme stability, and it would be surprising if in many very stable enzymes an increase in hydrophobicity could not be detected. Examples of increased enzyme stability caused by other weak interactions are also known [30,41,44].…”

N‐ and C‐capping preferences for all 20 amino acids in α‐helical peptides

“…The observed distributions of amino acids for these sites in protein crystal structures is quite different for preferences for interior positions (Richardson & Richardson, 1988). The importance of capping for helix structure and stability has been studied in peptide (Nicholson et al, 1988(Nicholson et al, , 1991Fairman et al, 1989;Bruch et al, 1991;Lyu et al, 1992Lyu et al, , 1993Chakrabartty et al, 1993a; 1326Forood et al, 1993, 1994Heinz et al, 1993;Regan, 1993;Doig et al, 1994;Zhou et al, 1994aZhou et al, , 1994b and protein systems (Serran0 & Fersht, 1989;Lecomte& Moore, 1991;Bell et al, 1992; Serrano et al, 1992b;Harper & Rose, 1993;Kaarsholm et al, 1993; Zhukovsky et al, 1994).Although several groups have measured the free energy changes that arise when certain residues are substituted at capping positions (see below), many amino acids have not been investigated at all. In this paper, we determine the intrinsic preferences for both N-and C-cap positions in our a-helical model peptide for all 20 naturally occurring amino acids in various charged states, plus the N-terminal acetyl and C-terminal amide groups.…”