title: Contribution of a single-turn alpha-helix to the conformational stability and activity of the alkaline proteinase inhibitor of Pseudomonas aeruginosa.
abstract: The alkaline proteinase inhibitor of Pseudomonas aeruginosa (APRin), a high-affinity inhibitor of the serralysin family of bacterial metalloproteinases, is folded into an eight-stranded beta-barrel with an N-terminal trunk linked to the barrel by a single-turn alpha-helix (helix A, residues 8-11). We show here that deletion or modification of helix A decreases the conformational stability of APRin as assessed by thermal and chemical denaturation with guanidinium chloride (GdmCl). The apparent melting temperature T(m) of the wild-type protein was 81.5 degrees C at pH 7.1 as assessed by circular dichroism and 87.5 degrees C by differential scanning calorimetry. Reduction of the single disulfide bond of APRin decreased T(m) by approximately 18 degrees C, while deletion of residues 6-10 or 1-10 lowered T(m) by approximately 8 and approximately 14 degrees C, respectively. DeltaG(u) as assessed by chemical denaturation was 7.2 kcal mol(-)(1) at 25 degrees C for wild-type APRin and was decreased by 3.4, 2.4, and 2.6 kcal mol(-)(1) by disulfide reduction, deletion of residues 6-10, and deletion of residues 1-10, respectively. In contrast, deletion of residues 1-5 had no significant effect on either T(m) or DeltaG(u). Substitution of five helix-breaking Gly or Pro residues in positions 6-10 as well as disruption of hydrogen bonds involving residues within helix A (mutants Asp10Pro and Trp15Phe) also decreased T(m) and DeltaG(u). The data suggest that a hydrogen-bonding network involving Leu11 in helix A and Trp15 located at the top of the barrel may prevent access of solvent to the interior of the barrel. Disruption of the helix could facilitate solvation of the nonpolar interior of the barrel, thereby destabilizing its folded structure. Kinetic studies with single amino acid mutants in helix A indicate that it modulates the affinity of APRin for APR primarily by influencing the dissociation rate of the inhibitor from the complex.
authors.affiliation: Department of Biochemistry & Molecular Biology, James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, Kentucky 40292, USA. rdgray@louisville.edu
authors.forename: Robert D
authors.initials: RD
authors.lastname: Gray
authors.forename: John O
authors.initials: JO
authors.lastname: Trent
mesh_terms: D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003198:Computer Simulation; D004220:Disulfides / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics; D013816:Thermodynamics
chemical_list: D004220:Disulfides; D010455:Peptides; D011480:Protease Inhibitors; C038356:microbial alkaline proteinase inhibitor; D019791:Guanidine