Recombinant Escherichia coli Elongation factor Ts (tsf)

What is the structural organization of E. coli Elongation Factor Ts?

E. coli Elongation Factor Ts (EF-Ts) functions as a guanine nucleotide-exchange factor for Elongation Factor Tu (EF-Tu). The protein contains a distinctive antiparallel coiled-coil motif that protrudes from the main structure and mediates dimerization in crystal structures. The functional core of EF-Ts consists of multiple domains including the N-terminal domain and Subdomain N, which play critical roles in its interaction with EF-Tu. The protein is encoded by a single gene (tsf) located in the rpsB-tsf operon of the E. coli chromosome .

How does the coiled-coil motif in EF-Ts contribute to its function?

The coiled-coil motif in E. coli EF-Ts plays a significant role in the protein's ability to compete with guanine nucleotides for binding to EF-Tu. Deletion experiments have shown that removing this motif substantially reduces the concentration of guanine nucleotides (GDP and GTP) required to dissociate the EF-Tu–EF-Ts complex by at least two orders of magnitude compared to the wild-type complex. While deletion of this motif only partially reduces EF-Ts's ability to stimulate guanine nucleotide exchange in EF-Tu, it significantly alters the competition between EF-Ts and kirromycin (an antibiotic) for binding to EF-Tu .

Which domains are most critical for EF-Ts interaction with EF-Tu?

Studies using chimeric proteins between E. coli EF-Ts and mammalian mitochondrial EF-Ts (EF-Tsmt) have revealed that Subdomain N of the core is particularly critical for strong binding to EF-Tu. When this subdomain from E. coli EF-Ts is replaced with the corresponding region from EF-Tsmt, the chimeric protein binds to E. coli EF-Tu approximately 25-fold more tightly than wild-type E. coli EF-Ts. The N-terminal domain also contributes to binding strength, as replacing the N-terminal domain of E. coli EF-Ts with that of EF-Tsmt increases its binding to E. coli EF-Tu 2-3-fold .

What is the precise role of EF-Ts in the elongation cycle of protein synthesis?

EF-Ts serves as the guanine nucleotide exchange factor for EF-Tu, which is essential for delivering aminoacyl-tRNA (aa-tRNA) to the ribosome during protein synthesis. EF-Tu has approximately 60-fold higher affinity for GDP over GTP and exhibits slow spontaneous nucleotide exchange. EF-Ts catalyzes the exchange of GDP for GTP on EF-Tu, thereby regenerating active EF-Tu·GTP that can form ternary complexes with aa-tRNAs. This nucleotide exchange activity is crucial for maintaining adequate levels of functional ternary complexes in the cell, making EF-Ts essential for cellular growth and normal rates of protein synthesis .

How does EF-Ts regulate ternary complex formation and stability?

Recent research has revealed that EF-Ts directly facilitates both the formation and dissociation of the EF-Tu·GTP·aa-tRNA ternary complex. EF-Ts accelerates a nucleotide-dependent, rate-determining conformational change in EF-Tu that controls both processes. Unexpectedly, EF-Ts attenuates the affinity of EF-Tu for GTP and destabilizes ternary complex in the presence of non-hydrolyzable GTP analogs. This suggests that EF-Ts plays an unanticipated regulatory role in controlling the abundance and stability of ternary complexes, contributing to rapid and faithful protein synthesis .

What happens to translation when EF-Ts function is impaired?

When EF-Ts function is compromised, such as through deletion of the coiled-coil motif, bacterial growth rates can be reduced to 70–95% of wild-type, depending on growth conditions. Interestingly, such mutations can affect how cells respond to amino acid starvation, with mutant strains sensing starvation and synthesizing stress signaling nucleotides like guanosine 5′-diphosphate 3′-diphosphate (ppGpp) at lower cell densities than wild-type strains. This indicates that EF-Ts plays a role in translational regulation during stress conditions .

What expression systems are optimal for producing recombinant E. coli EF-Ts?

Recombinant E. coli EF-Ts can be efficiently expressed in E. coli expression systems using standard vectors with inducible promoters such as T7. The protein typically expresses in soluble form due to its intrinsic high folding efficiency. For experimental studies, the tsf gene can be amplified from E. coli genomic DNA and cloned into expression vectors that provide appropriate tags for purification (such as His-tag or chitin-binding domain). Expression can be optimized using E. coli BL21(DE3) or similar strains under controlled induction conditions to maximize yield while maintaining proper folding .

What methodologies can be used to study EF-Ts interactions with EF-Tu?

Several methodologies are effective for studying EF-Ts:EF-Tu interactions:

• Guanine nucleotide exchange assays using radiolabeled GDP or fluorescent GDP analogs

• Surface plasmon resonance (SPR) to determine binding kinetics and affinity constants

• Isothermal titration calorimetry (ITC) for thermodynamic parameters of interaction

• Chimeric protein construction between different species' EF-Ts (e.g., E. coli and mitochondrial) to identify critical interaction domains

• Deletion mutations to examine the role of specific motifs like the coiled-coil domain

• Two-dimensional electrophoresis to analyze expression levels under different conditions

• Crystal structure analysis of EF-Tu·EF-Ts complexes

These approaches have revealed that bovine mitochondrial EF-Ts forms a complex with E. coli EF-Tu with an association constant of 8.6 × 10^10, which is 100-fold stronger than the binding constant for E. coli EF-Tu·Ts complex formation .

How effective is EF-Ts as a fusion partner for enhancing protein solubility?

EF-Ts has been identified as an effective solubility-enhancing fusion partner for heterologous protein expression in E. coli. Proteome analysis revealed that EF-Ts expression increases 1.61-fold under protein denaturation stress conditions while many other host proteins aggregate or decrease in synthesis. When used as an N-terminal fusion partner, EF-Ts has been shown to dramatically improve the soluble expression of various heterologous proteins that would otherwise form inclusion bodies when directly expressed. This makes EF-Ts a valuable biotechnological tool for producing soluble, active recombinant proteins .

What mechanisms explain EF-Ts's ability to enhance protein solubility?

The solubility-enhancing effect of EF-Ts appears to be due to its intrinsic high folding efficiency. When fused to the N-terminus of target proteins, EF-Ts is thought to play a critical role in sequestering interactive surfaces of heterologous proteins from nonspecific protein-protein interactions that typically lead to inclusion body formation. This chaperone-like activity helps maintain the target protein in a soluble state during expression and folding. The effectiveness of EF-Ts has been demonstrated with bacterial cutinase, producing biologically active enzyme that could be valuable for biotechnology applications .

What are the advantages of using EF-Ts for stress-induced protein expression?

EF-Ts shows increased expression under protein denaturation stress conditions (e.g., in the presence of guanidine hydrochloride), making it particularly useful for stress-induced expression systems. While many E. coli proteins aggregate or show decreased synthesis under stress (34 out of 699 soluble proteins disappear and 63 decrease by over 2.5-fold), EF-Ts expression increases by 1.61-fold. This natural stress response can be leveraged in biotechnological applications to optimize production of difficult-to-express proteins by creating expression systems where EF-Ts fusion proteins are preferentially expressed under controlled stress conditions .

How do bacterial and mitochondrial EF-Ts differ in structure and function?

Bacterial (E. coli) and mammalian mitochondrial EF-Ts (EF-Tsmt) show significant differences in their interactions with EF-Tu. While bovine EF-Tsmt can stimulate the activity of E. coli EF-Tu, E. coli EF-Ts is unable to stimulate mitochondrial EF-Tu. The binding affinity also differs dramatically, with EF-Tsmt forming a complex with E. coli EF-Tu that is 100-fold stronger than the E. coli EF-Tu·Ts complex. These functional differences can be primarily attributed to Subdomain N of the core, as replacing this region in E. coli EF-Ts with the corresponding region from EF-Tsmt increases binding to E. coli EF-Tu by approximately 25-fold .

How can chimeric constructs inform our understanding of EF-Ts function?

Chimeric constructs between E. coli EF-Ts and mammalian mitochondrial EF-Ts have been instrumental in mapping functional domains. Studies have shown that:

• Replacing the N-terminal domain of E. coli EF-Ts with that of EF-Tsmt increases binding to E. coli EF-Tu 2-3-fold

• Replacing the N-terminal domain of EF-Tsmt with the corresponding region of E. coli EF-Ts decreases binding to E. coli EF-Tu approximately 4-5-fold

• A chimera consisting of the C-terminal half of E. coli EF-Ts and the N-terminal half of EF-Tsmt binds to E. coli EF-Tu as strongly as EF-Tsmt

• A chimera in which Subdomain N of E. coli EF-Ts is replaced by the corresponding region of EF-Tsmt binds E. coli EF-Tu approximately 25-fold more tightly than E. coli EF-Ts

These findings localize the higher interaction strength between EF-Tsmt and EF-Tu primarily to Subdomain N, providing valuable insights into domain-specific functions .

What are the functional consequences of deleting the coiled-coil motif in EF-Ts?

Deletion of the coiled-coil motif from the E. coli EF-Ts genome produces several significant functional consequences:

• Reduced growth rate: The mutant strain grows at 70–95% of the wild-type rate, depending on growth conditions

• Altered stress response: The mutant strain senses amino acid starvation and synthesizes stress signaling nucleotides (ppGpp and pppGpp) at lower cell density than wild-type

• Partial reduction in nucleotide exchange activity: The deletion only partially reduces EF-Ts's ability to stimulate guanine nucleotide exchange in EF-Tu

• Dramatically altered nucleotide binding competition: The concentration of guanine nucleotides required to dissociate the mutant EF-Tu–EF-Ts complex is at least two orders of magnitude lower than for the wild-type complex

• Changed antibiotic interaction: The deletion alters the competition between EF-Ts and kirromycin for binding to EF-Tu

These findings demonstrate that the coiled-coil motif plays a significant role in EF-Ts's ability to compete with guanine nucleotides for binding to EF-Tu .

How can EF-Ts be engineered to optimize protein expression systems?

Based on the available research, several engineering strategies could optimize EF-Ts for protein expression systems:

• Domain optimization: Creating chimeric constructs that incorporate the stronger-binding domains from mitochondrial EF-Ts while maintaining compatibility with bacterial expression systems

• Coiled-coil modifications: Engineering the coiled-coil domain to fine-tune interaction with EF-Tu and optimize nucleotide exchange rates

• Fusion protein design: Developing optimized linker sequences between EF-Ts and target proteins to maximize solubility enhancement while minimizing impact on target protein function

• Stress-response elements: Incorporating regulatory elements that increase EF-Ts fusion protein expression under specific stress conditions

These approaches could lead to expression systems with enhanced production of soluble, active recombinant proteins, particularly for difficult-to-express targets that typically form inclusion bodies .