Circular dichroic measurements of the host peptide acetyl-Y(EAAAK)3A-amide were obtained in solutions of increasing ionic strength at pH 7.0 and 0 degree C. The changes observed in the dichroic spectra are characteristic for a two-state helix/coil transition. The mean residue ellipticity at 222 nm exhibits a curvilinear dependence on ionic strength which becomes linear at ionic strengths greater than 1 M. The slope of the linear portion is assumed to represent the lyotropic character of the salt, and its extrapolated intercept is assumed to represent the mean residue ellipticity of the peptide solution freed from both electrostatic and lyotropic contributions which affect the helical stability of the host peptide. An extrapolated mean residue ellipticity value was obtained for each host peptide having a different amino acid guest residue at position 9 in the peptide sequence. These values were used to calculate a propagation parameter, s, for each residue using the Lifson-Roig algorithm for peptide helical content and assuming a common nucleation parameter of 0.003. The ability of these minimally determined residue parameters to predict the helical content of a variety of peptides is encouraging. Estimates were also made of the delta G values for the electrostatic interactions within the host peptide and for the additional interactions generated by ionic guest residues.

In common globular proteins, the native form is in its most stable state. In contrast, each native form exists in a metastable state in inhibitory serpins (serine protease inhibitors) and some viral membrane fusion proteins. Metastability in these proteins is critical to their biological functions. Mutational analyses and structural examination have previously revealed unusual interactions, such as side-chain overpacking, buried polar groups, and cavities as the structural basis of the native metastability. However, the mechanism by which these structural defects regulate protein functions has not been elucidated. We report here characterization of cavity-filling mutations of ␣1-antitrypsin, a prototype serpin. Conformational stability of the molecule increased linearly with the van der Waals volume of the side chains. Increasing conformational stability is correlated with decreasing inhibitory activity. Moreover, the activity loss appears to correlate with the decrease in the rate of the conformational switch during complex formation with a target protease. These results strongly suggest that the native metastability of proteins is indeed a structural design that regulates protein functions.T he native forms of most proteins are thermodynamically the most stable state (1). However, the native forms of some proteins are metastable: typical examples are the strained native structure of plasma serpins (serine protease inhibitors) (2), the spring-loaded structure of the membrane fusion protein of inf luenza virus (3, 4), heat shock transcription factors (5), and possibly the surface glycoprotein of human immunodeficiency virus (HIV) (6). Metastability in these proteins is considered to be an important mechanism for regulating their biological functions (2-8). The native strain of serpins is crucial to their physiological function, such as protease inhibition (2, 7), hormone delivery (9), Alzheimer filament assembly (10), and extracellular matrix remodeling (11). The inhibitory serpins include ␣ 1 -antitrypsin (␣ 1 AT), ␣ 1 -antichymotrypsin, antithrombin-III, plasminogen activator inhibitor-1, C1-inhibitor, and antiplasmin, which regulate processes such as inf lammation, coagulation, fibrinolysis, and complement activation (2). The serpin structure is composed of three ␤-sheets and several ␣-helices, and the reactive center loop is exposed at one end of the molecule for protease binding (Fig. 1). Upon binding a target protease, the reactive center loop of inhibitory serpins is thought to be inserted into the major ␤-sheet, A sheet, to form a very stable complex between the inhibitor and the protease (12). Because the metastable native form has an advantage of facile conversion into an alternative more stable conformation, it is conceivable that the native metastability of serpins is used for the facile conformational change during the complex formation.To understand the structural basis and functional role of the native metastability, we have characterized stabilizing amino acid substitutions of ␣ 1 AT, a prototyp...

The peptide acetylYEAAAKEARAKEAAAKAamide exhibits the dichroic features characteristic of a monomeric helix/coil transition in aqueous solution. Nineteen variants of this peptide each containing a different residue at position 9 were prepared by solid-phase peptide synthesis and purified by reversed-phase chromatography. The thermal dependence of the far-ultraviolet dichroic spectrum of each of these peptides except that containing proline is characteristic for an alpha-helix/coil transition. The relative stability of the helical forms of these peptides does not correlate with the preference of the variable amino acid to occupy a middle position in a protein helix. It is likely that the specific interactions of the variable residue with its local environment obscure any inherent preference of the residue to reside in an alpha-helix.

The mean residue ellipticity of the helical host peptide, acetyl-YEAAAKEAXAKEAAAKA-amide containing guest residues at position X, was measured as a function of pH and ionic strength at 0 degree C. Changes in ellipticity at 222 nm were interpreted in terms of a two-state helix/coil transition of a monomeric peptide. Variable pH measurements in low concentrations of KCl defined changes in helix stability resulting from the ionization of each guest residue. Variable [KCl] measurements at fixed pH generated ellipticity values for the neutral and ionic forms of each guest residue free of electrostatic and lyotropic contributions. These ellipticity values were used to calculate a helix propagation parameter for each form of a guest residue using the Lifson-Roig algorithm and assuming a universal nucleation parameter. In all cases, the propagation parameter of a residue is either unaffected or decreased by ionization of its side chain.