The exceptionally high density of the protein interior shown here implies that packing forces play a more important role in protein stability than has been believed hitherto.

The structural basis for the stability of N termini of helices has been analyzed by thermodynamic and crystallographic studies ofthree suitably engineered mutants of the barley chymotrpsin inhibitor 2 with Ser, Gly, or Ala at the N-cap position (residue 31). Each mutant has a well-organized shell of hydration of the terminal NH groups of the helix. The three structures are virtually superimposable (rms separations for all atoms, induding the common water molecules, are 0.15-0.17 A) and show neither changes in conformation at the site of substitution nor changes in the crystal packing. The only changes on going from Ser-31 to Ala-31 to Gly-31 are in the position of a water molecule AAGAla--,Gly =1.82 (±0.25) -(0.046 ± 0.013)A&AHP -(0.19 ± 0.03)AAHB (kcal-mol-1), [1] (modified from and recalculated as described in refs. 6 and 8, using latest unpublished data from this laboratory on barnase; 1 kcal = 4.18 kJ). The term in AAHP is probably reliable because of commonly found relationships between free energies of transfer of hydrophobic surface groups and surface (9), but the term in AAHB lacks precedence and requires further research before its importance can be generalized.The stabilities and crystal structures of six mutants at the N-cap residue (Thr-59) ofthe long helix ofT4 phage lysozyme have been determined. The relative stabilities are Thr Ser Asp > Asn > Gly Ala Val (10). In none of these structures, however, was a new water molecule seen to replace the hydrogen-bonding function of the -t-hydroxyl in the wild-type Thr variant. Bell et aL (10) disputed the hypothesis of Serrano et al. (6,8) that Gly and Ala are better helix-capping residues than Val because a smaller residue allows better hydration at the end of the helix. However, Serrano et al. (6) noted that the side chain of residue Asp-61 in T4 lysozyme moves to become a surrogate N-cap on mutation ofThr-51 to Ala, Gly, and Val, so lessening the need for external solvation.In the present study, we report the energetics and crystal structures of a series of mutants at the N-cap, position 31, of the barley chymotrypsin inhibitor 2 (CI2); Ser-31, Ala, and Ser-31 --Gly. As this region of the protein is disordered in the crystals of wild-type protein (11, 12), we have constructed a pseudo-wild-type (pseudo-wt) protein (Glu-33 -* Ala and Glu-34 -* Ala) which is suitable for crystallographic study and have used this as the reference protein. These mutations also remove the possibility of the side chain ofGlu-33 becoming a surrogate N-cap. The protein was also truncated by the deletion of the N-terminal tail of 20 residues that is disordered in both the crystal and solution structures (11)(12)(13) to give a 64-residue fragment that is very suitable for x-ray studies.Abbreviations: C12, chymotrypsin inhibitor 2; pseudo-wt, pseudowild-type.