J. Mol. Biol. (1996) 263, 390–395 COMMUNICATION The -Helix of Ribonuclease T 1 as an Independent Stability Unit: Direct Comparison of Peptide and Protein Stability Jeffrey K. Myers 1,2 , Jeni S. Smith 1 , C. Nick Pace 1,2 and J. Martin Scholtz 1,2 * 1 Department of Medical Measurements of the change in conformational stability, (G), upon mutation of two acidic residues at the C terminus of the helix of Biochemistry and Genetics and 2 Department of ribonuclease T 1 have recently been reported. Here, we investigate peptides based on the sequence of the helix with the same mutations: Glu28 Biochemistry and Biophysics Center for Macromolecular replaced with Gln, Asp29 replaced with Asn, and the double mutant. In Design, Texas A&M addition, the mutant Lys25 to Gln was studied. Changes in helix content University, College Station of the peptides with pH confirm the conclusion found in the intact protein, TX 77843, USA that the charged forms of the acidic residues destabilize the protein by destabilizing the helix. The pH-dependence of the change in confor- mational free energy for the peptides and mutant proteins show fair correspondence for D29N and the double mutant. The mutants E28Q and K25Q, on the other hand, give striking agreement between the protein and peptide systems. This agreement suggests that the helix of ribonuclease T 1 behaves as an independently stabilized structural unit of the intact protein and that stabilization of the helical form of the peptide is mirrored in the protein.  1996 Academic Press Limited Keywords: protein stability; electrostatic interactions; -helix; peptide; *Corresponding author independent stability unit The many different non-covalent interactions stabilizing globular proteins have been the object of study for many years. The advent of site-directed mutagenesis provided a powerful tool: the change in conformational stability caused by an amino acid replacement gives an estimate of the contributions to stability of the particular interactions altered by the mutation. This popular approach has provided useful information, particularly on the contri- butions of electrostatic interactions, hydrophobic interactions and hydrogen bonds (Pace, 1992; Fersht & Serrano, 1993; Matthews, 1995; Myers & Pace, 1996). Another approach is to use structured peptides to model interactions in proteins. Peptides are less complicated systems in which factors stabilizing secondary structure can be studied without the complexity of tertiary interactions. Here, we provide a direct comparison of changes in stability brought about by mutations in an intact protein and a helical peptide derived from that protein. Our results are consistent with the hierarchical or framework model for protein folding and stability (Crippen, 1978; Rose, 1979; Kim & Baldwin, 1982, 1990), and we find that the helix of RNase T 1 is an independent stability unit of the intact protein. Small peptides that adopt an -helical structure in aqueous solution have been of particular interest in testing the hierarchical nature of protein stability (Dyson et al ., 1992; Scholtz & Baldwin, 1992, 1995; Chakrabartty & Baldwin, 1995). Alanine-based helical peptides, in particular, have provided a wealth of information on the thermodynamics of helix formation (Scholtz et al ., 1991a), interactions between side-chains (Scholtz et al ., 1993; Huyghues- Despointes et al ., 1995), and the intrinsic helical propensities of the amino acids (Park et al ., 1993; Chakrabartty et al ., 1994). In the course of these and other studies on helical peptides, it was realized that positions at the ends of helices are different from those in the middle. The ends of helices have two important and distinct characteristics. First, as dictated by the (i , i + 4) hydrogen-bonding pattern of the helix, the first four and the last four resi- dues have unsatisfied backbone hydrogen-bonding groups. It has been shown in both peptides and 0022–2836/96/430390–06 $25.00/0  1996 Academic Press Limited