The disulfide bond signal sequence (DsbAss) protein is characterized as an important virulence factor in gram-negative bacteria. This study aimed to analyze the “alanine” alteration in the hydrophobic (H) region of DsbAss and to understand the conformational DsbAss alteration(s) inside the fifty-four homolog (Ffh)-binding groove which were revealed to be crucial for translocation of ovine growth hormone (OGH) to the periplasmic space in Escherichia coli via the secretory (Sec) pathway. An experimental design was used to explore the hydrophobicity and alteration of alanine (Ala) to isoleucine (Ile) in the tripartite structure of DsbAss. As a result, two DsbAss mutants (Ala at positions -11 and -13) with same hydrophobicity of 1.539 led to the conflicting translocation of the active OGH gene. We performed molecular dynamics (MD) simulations and molecular mechanics generalized born surface area (MM-GBSA) binding free energy calculations to examine the interaction energetic and dynamic aspects of DsbAss/signal repetition particle 54 (SRP54) binding, which has a principle role in Escherichia coli Sec pathways. Although both DsbAss mutants retained helicity, the MD simulation analysis evidenced that altering Ala-13 changed the orientation of the signal peptide in the Ffh M binding domain groove, favored more stable interaction energies (MM-GBSA ΔG_total = −140.62 kcal mol⁻¹), and hampered the process of OGH translocation, while Ala-11 pointed outward due to unstable conformation and less binding energy (ΔG_total = −124.24 kcal mol⁻¹). Here we report the dynamic behavior of change of “alanine” in the H-domain of DsbAss which affects the process of translocation of OGH, where MD simulation and MM-GBSA can be useful initial tools to investigate the virulence of bacteria.

Lupanine hydroxylase (LH), a quinohaemoprotein, catabolizes lupanine and possesses four cysteine (Cys) residues; two associated with a cytochrome c mo

A chimeric mammalian globular cytochrome b₅ fused to Escherichia coli alkaline phosphatase signal sequence (SS) was used as a model probe to investigate the influence of substituting each one of the standard 20 amino acids at its N‐terminus on the Sec‐dependent export of the precursor to the periplasmic space of E. coli. Substituting the native Met⁺¹ of the passenger protein flanking the SS with any one of the remaining 19 amino acids introduced significant changes in the export of cytochrome b₅ without jamming the Sec‐dependent translocon. Acidic and hydrophilic residues proved to be the most efficient promoters of export. Small, nonbulky and basic residues yielded intermediate levels of the hemoprotein export. Replacement with a Cys⁺¹ residue generated significant quantities of both monomeric and disulfide‐linked dimeric forms. However, bulky, aromatic and hydrophobic residues caused a significant decline in the rates of secretion. In expectation with their absences in the natural periplasmically secreted proteins, Pro and Ile‐tagged cytochrome b₅ precursors failed to generate any detectable secreted recombinant products. Although Ala, amongst the native E. coli periplasmic proteins, is the preferred X⁺¹ residue with an occurrence of 50% frequency, it proved half as effective in promoting export when inserted proximally to the SS of cytochrome b₅. The mechanisms involved for these export variations are discussed. The findings will prove beneficial for high‐level generation of recombinant proteins by secretory means for pharmaceutical and related biotechnological applications.