title: Differences in the structural stability and cooperativity between monomeric variants of natural and de novo Cro proteins revealed by high-pressure Fourier transform infrared spectroscopy.
abstract: It is widely accepted that pressure affects the structure and dynamics of proteins; however, the underlying mechanism remains unresolved. Our previous studies have investigated the effects of pressure on fundamental secondary structural elements using model peptides, because these peptides represent a basis for understanding the effects of pressure on more complex structures. This study targeted monomeric variants of naturally occurring bacteriophage λ Cro (natural Cro) and de novo designed λ Cro (SN4m), which are α + β proteins. The sequence of SN4m is 75% different from that of natural Cro, but the structures are almost identical. Consequently, a comparison of the folding properties of these proteins is of interest. Pressure- and temperature-variable Fourier transform infrared spectroscopic analyses revealed that the α-helices and β-sheets of natural Cro are cooperatively and reversibly unfolded by pressure and temperature, whereas those of SN4m are not cooperatively unfolded by pressure; i.e., the α-helices of SN4m unfold at significantly higher pressures than the β-sheets and irreversibly unfold with increases in temperature. The higher unfolding pressure for the α-helices of SN4m indicates the presence of an intermediate structure of SN4m that does not retain β-sheet structure but does preserve the α-helices. These results demonstrate that the α-helices of natural Cro are stabilized by global tertiary contacts among the α-helices and the β-sheets, whereas the α-helices of SN4m are stabilized by local tertiary contacts between the α-helices.
authors.affiliation: Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Imamura
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Isogai
authors.forename: Minoru
authors.initials: M
authors.lastname: Kato
mesh_terms: D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000737:chemistry*; D002942:Circular Dichroism; D048168:Escherichia coli K12 / Q000737:chemistry / Q000235:genetics / Q000821:virology; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D012097:Repressor Proteins / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013816:Thermodynamics; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry*
chemical_list: D012097:Repressor Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins