All the amino acids are found in α-helices, but glycine and proline are uncommon, as they destabilize the α-helix. Glycine is exempt from many steric constraints because it lacks a β carbon. Because glycine residues have more conformational freedom than other residues, glycine favors the unfolded conformation over the helix conformation. Proline, on the other hand, is too rigid. Steric crowding between the 5-membered ring of proline residue in the middle of α-helix and the preceeding residue causes a kink the helix. However, it is worth noting that about half of the kinked α-helices do not have prolines.
For two decades after the crystallographic structure of myoglobin was solved at atomic resolution, proline residues were never seen in the middle of an α-helix. Now that there are over 30,000 protein structures in the Protein Data Bank, it is clear that proline residues are present in α-helices, where they often play important roles in the structure and function of the protein.
Part I: Proline Exposes Two Carbonyl Oxygens
Let's look at another α-helix; it comes from the crystal structure of actin at high resolution and contains eight residues. Actin is found in all eukaryotic cells and is frequently the most abundant protein in these cells. Actin filaments are an important part of the cytoskeleton. Actin is also found in combination with myosin in muscle.
Note that there are only three hydrogen bonds in this α-helix. How many should a helix with eight residues have? Suggest an explanation for the fact that there are only three hydrogen bonds.
space-filling model. All side chains except the side chain of proline are omitted from the model.
Proline lacks an amide proton when found within proteins. This precludes hydrogen bonding between it and hydrogen bond acceptors, and thus often restricts the Pro residue to the first four positions of an α helix. Positioning a proline after position four, as in the helix you are viewing, results in the exposure of two backbone carbonyl oxygen atoms. These exposed atoms often serve as important sites for ligand binding.
exposed carbonyl oxygen atoms.
Complete model showing all side chains. Is this helix capped at the N-terminus? At the C-terminus?
Part II: Proline Kinks
The helix from actin is fairly straight. In many instances, however, proline induces a kink in the helix; this often aids in the packing of long helices in both cytosolic and transmembrane proteins.
The proline residue lacks an amide proton. This precludes hydrogen bonding between it and hydrogen bond acceptors, and thus often restricts the proline residue to the first four positions of an α-helix. Proline has "helix-like" backbone dihedral angles that help to initiate helix folding.
The presence of a proline in the interior of an α-helix induces a kink in the helical axis. Proline kinked helices are almost exclusively long helices (>4 turns); this often aids in the packing of long helices in a protein.
FIS (Factor for Inversion Stimulation) protein, a DNA-binding protein from E. coli. There are two identical subunits in the molecule. The first 25 N-terminal residues are disordered and are not observed in the electron density map.
R. C. Johnson investigated the influence of proline on bending of the α-helix by directed replacement of the proline residue located in the middle of the long B helix of the FIS protein with alanine. The Pro61Ala variant has the same Tm as the wild type and is fully active in stimulating Hin-mediated DNA inversion.
The crystal structure of the Pro61Ala variant is essentially identical to the wild-type protein, consistent with its full activity. One of the α-helices, the B helix, is kinked in the wild type FIS protein by 20 degrees. This kink was previously assumed to be caused solely by the presence of Proline-61 in the helix. However, the B helix is still kinked by 16 degrees when Proline-61 is replaced by alanine.
α-Helices in the monomer. A backbone model makes it easier to see the kink at .
Since kinking of the B helix occurs without proline at position 61, we can assume that the geometry of the helix is determine by local interactions with other parts of the protein, not by the rigid ring of Pro 61.