Recombinant Shewanella putrefaciens ATP synthase subunit b (atpF)

Shewanella putrefaciens Characteristics

Shewanella putrefaciens is a Gram-negative bacterium known for causing seafood spoilage, particularly under low-temperature conditions. This organism readily forms biofilms to enhance its survival in challenging environments . Notably, comparative transcriptome analyses have revealed that biofilms formed by S. putrefaciens WS13 at low temperatures (4°C) demonstrate larger biomass and tighter structure compared to those formed at optimal growth temperatures (30°C) . This cold adaptation capability makes S. putrefaciens a significant concern in food preservation.

ATP Synthase Complex Overview

ATP synthase, also known as F-type ATPase, is a multi-subunit enzyme complex essential for energy metabolism in all living organisms. The complex consists of two primary sectors: the membrane-embedded F₀ sector and the catalytic F₁ sector. The ATP synthase subunit b (atpF) is a critical component of the F₀ sector and serves as part of the peripheral stalk that connects the F₀ and F₁ sectors. This arrangement is crucial for the proper functioning of the entire complex in ATP synthesis during oxidative phosphorylation.

Protein Identity and Sequence

The ATP synthase subunit b (atpF) from Shewanella putrefaciens is identified in protein databases with the UniProt accession number A4YCI2 . The full-length protein consists of 156 amino acids with the following sequence:

MSINATLLGQAISFALFVWFCIKFVWPPLMNAIEERQKKIADGLADAGRAAKDLELAQAKATEQLKEAKVTANEIIEQANKRKAQIVEEAKAEADAERAKIIAQGKAEIENERSRVKDDLRKQVAALAVLGAERILERSIDQAAHSDIVDKLVAEI

This protein is often referred to by several synonyms, including ATP synthase subunit b, ATP synthase F(0) sector subunit b, ATPase subunit I, F-type ATPase subunit b, and F-ATPase subunit b .

Recombinant Protein Properties

The commercially available recombinant form of the protein typically includes the following specifications:

This data demonstrates the high quality and defined characteristics of the recombinant protein preparation available for research purposes .

Role in Energy Production

ATP synthase subunit b (atpF) serves as an integral component of the ATP synthase complex, which is responsible for ATP production in the cell. The subunit b functions primarily as part of the peripheral stalk or stator that prevents rotation of the F₁ catalytic sector during ATP synthesis. This structural stabilization is essential for the proper functioning of the enzyme, enabling the central rotor to turn relative to the stationary catalytic sites, thereby facilitating the conformational changes necessary for ATP synthesis.

Adaptation to Environmental Conditions

In the context of Shewanella putrefaciens' ability to thrive in cold environments, the ATP synthase complex, including subunit b, may undergo specific adaptations to maintain energy production efficiency at low temperatures. Comparative transcriptome analysis of S. putrefaciens has identified oxidative phosphorylation as one of the significantly changed metabolic pathways under cold stress (4°C) . This suggests potential modifications in ATP synthase components, including the atpF subunit, to optimize energy production under refrigeration conditions.

Expression Systems and Methodology

Recombinant Shewanella putrefaciens ATP synthase subunit b (atpF) is typically expressed in Escherichia coli expression systems . The gene encoding the full-length protein (1-156 amino acids) is cloned and expressed with an N-terminal histidine tag to facilitate purification and detection. This approach is similar to the expression methods used for other ATP synthase subunits from S. putrefaciens, such as the delta subunit (atpH), which has been expressed using baculovirus systems .

Purification Process

Following expression, the recombinant protein is purified to a high degree of homogeneity using immobilized metal affinity chromatography (IMAC), taking advantage of the histidine tag. The purification results in a product with greater than 90% purity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . This level of purity ensures the reliability of subsequent biochemical and structural studies.

Quality Assessment

The quality of the purified recombinant protein is typically assessed using SDS-PAGE to confirm size, purity, and integrity. Additional analytical methods may include mass spectrometry for precise molecular weight determination and functional assays to verify the protein's biological activity or binding properties.

Cold Adaptation Research

Given S. putrefaciens' significant role in seafood spoilage under refrigeration conditions, research on its ATP synthase components, including the atpF subunit, provides insights into cold adaptation mechanisms. Transcriptome analysis has revealed that oxidative phosphorylation pathways, which include ATP synthase, undergo significant changes under cold stress . Understanding these adaptations could lead to improved food preservation strategies.

Comparative Studies with Other Bacterial Species

Comparative analysis of ATP synthase components across different bacterial species can reveal evolutionary adaptations to specific ecological niches. For instance, comparing the atpF subunit from S. putrefaciens with those from non-psychrotolerant bacteria might illuminate the structural features that contribute to cold adaptation.

Reconstitution Protocol

Prior to opening, it is recommended that the vial containing the lyophilized protein be briefly centrifuged to bring the contents to the bottom . The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, adding glycerol to a final concentration of 5-50% (typically 50%) is recommended before aliquoting and storing at -20°C or -80°C .