Recombinant Escherichia coli Inner membrane protein ygaP (ygaP)

What is the basic structure of the YgaP protein?

YgaP is a membrane protein with a molecular mass of 18.6 kDa, composed of 174 amino acid residues . Its structure consists of two main components: a cytoplasmic rhodanese domain (residues 1-118) and two transmembrane helices (residues 119-174) . According to topology prediction methods like TMHMM, the rhodanese domain resides in the cytoplasm, while residues 119-174 form the transmembrane region . In its native state, YgaP forms a homodimer with the transmembrane helices serving as the dimer interface . The N-terminal cytoplasmic domain contains a rhodanese-fold, consistent with its sulfurtransferase activity .

How does YgaP compare to other rhodanese domain proteins in E. coli?

E. coli encodes eight proteins containing rhodanese domains, but only three of these (GlpE, PspE, and YgaP) are composed of a single rhodanese domain . Among these three proteins, YgaP is unique as it is the only membrane-bound rhodanese domain protein in E. coli . While the crystal structure of sulfur-free and persulfide forms of GlpE and solution state NMR structure of PspE have been determined, YgaP provides a distinct research opportunity as an integral membrane sulfurtransferase .

What is the primary function of YgaP in bacterial cells?

The primary function of YgaP appears to be sulfurtransferase activity, specifically involved in the detoxification of cyanide (CN-) to the less toxic thiocyanate (SCN-) . Like other rhodanese family members, YgaP catalyzes a two-step reaction: first, it accepts a sulfur atom from a donor such as thiosulfate (S2O3-) to form an enzyme-persulfide intermediate at the catalytic cysteine residue (Cys-63); second, this intermediate reacts with cyanide ion to produce thiocyanate . The resulting conformational changes in both the rhodanese domain and transmembrane helices suggest that YgaP may also be involved in the export of thiocyanate across the membrane .

What structural changes occur in YgaP during catalysis?

During catalysis, YgaP undergoes significant conformational changes that have been observed using both NMR and EPR techniques. When YgaP binds to thiosulfate, several sulfur atoms can be added to the catalytic Cys-63 in a concentration-dependent manner . This process can be reversed by adding potassium cyanide. The catalytic reaction induces conformational changes in:

• The rhodanese domain - Affecting the active site geometry around Cys-63

• The transmembrane α-helices - Leading to tighter packing of the second transmembrane helices

These structural rearrangements appear to be functionally important, potentially creating a pathway for thiocyanate export across the membrane .

How was the tertiary structure of full-length YgaP determined?

The tertiary structure of full-length YgaP was determined through a combination of solution NMR and EPR-based approaches . The methodology involved:

• NMR studies: The solution NMR structure was determined in mixed micelles (1,2-diheptanoyl-sn-glycerol-3-phosphocholine/1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol)) . Sequential backbone assignment was obtained primarily from three-dimensional ct-TROSY-HNCA experiments, with ambiguities resolved using ct-TROSY-HN(CO)CA .

• EPR analysis: Systematic site-specific EPR analysis defined the helix-loop-helix secondary structure of the YgaP-TMD monomers using mobility, accessibility, and membrane immersion measurements . The tertiary folds of dimeric YgaP-TMD and full-length YgaP in detergent micelles were determined through inter- and intra-monomer distance mapping and rigid-body computation .

• Integrated approach: For identifying intermolecular NOEs between neighboring subunits, a mixed sample was prepared with a 1:1 ratio of 2H, 15N-labeled protein and unlabeled protein . Three-dimensional 15N-resolved 1H, 1H NOESY spectra were then recorded to identify interactions between monomers .

What is the significance of YgaP dimerization for its function?

YgaP forms a homodimer with the two transmembrane helices creating the dimer interface . This dimerization appears functionally significant for several reasons:

• Structural stability: The dimeric arrangement provides stability to the membrane-embedded portion of the protein

• Conformational coupling: The dimerization allows for communication between the cytoplasmic rhodanese domains through the transmembrane helices

• Thiocyanate export: EPR analysis shows that the two second transmembrane helices pack more tightly upon binding of SCN-, suggesting that dimerization creates a dynamic channel or pathway that facilitates the export of thiocyanate across the membrane

The dimeric structure thus appears to couple the catalytic activity of the rhodanese domains with the transport function of the transmembrane region, providing a potential mechanism for YgaP's role in cyanide detoxification .

What expression systems and purification protocols work best for recombinant YgaP?

Based on the available research, the following expression and purification approach has been successfully used for YgaP:

For researchers designing constructs, it should be noted that all YgaP preparations used in the referenced studies lacked the Met at the N terminus compared with the UniProt database entry, making Ala the first amino acid residue (numbered Ala-1) .

How can YgaP's sulfurtransferase activity be measured experimentally?

The enzymatic sulfur transfer activity of YgaP can be measured using the following methods:

• NMR titration experiments: A series of titrations with sodium thiosulfate and potassium cyanide, monitored by NMR spectroscopy . This approach allows for the observation of chemical shift changes associated with the addition of sulfur atoms to Cys-63 and subsequent reaction with cyanide.

• EPR measurements: EPR spectroscopy can be used to monitor conformational changes induced by substrate binding and catalysis . This is particularly useful for observing changes in the transmembrane helices upon SCN- binding.

• Enzymatic assay protocol:

  • Express and purify full-length YgaP or the N-terminal rhodanese domain only

  • Perform titrations with varying concentrations of sodium thiosulfate

  • Monitor the formation of the enzyme-persulfide intermediate

  • Add potassium cyanide and observe the reversal of the process

  • Measure the production of thiocyanate (SCN-)

These methods can provide insights into both the catalytic mechanism and the structural changes associated with YgaP's sulfurtransferase activity.

What NMR spectroscopy techniques are most effective for studying YgaP structure?

Several NMR techniques have proven effective for studying the structure of YgaP:

For best results, researchers should use 2H (∼50%), 13C, 15N-labeled YgaP to improve spectral quality, as T2 relaxation in 13C, 15N-labeled samples can cause severe signal loss in the transmembrane region .

What is the mechanism of YgaP's sulfurtransferase activity?

The mechanism of YgaP's sulfurtransferase activity involves a two-step reaction:

• First step: YgaP accepts a sulfur atom from thiosulfate (S2O3-) to form an enzyme-persulfide intermediate at the catalytic Cys-63 .

• Second step: This intermediate reacts with the cyanide ion to produce thiocyanate (SCN-) .

NMR and EPR data indicate that the addition of sulfur atoms to Cys-63 is thiosulfate concentration-dependent, with multiple sulfur atoms potentially being added . This process can be reversed by the addition of potassium cyanide . The catalytic reaction induces conformational changes in both the rhodanese domain and the transmembrane helices, suggesting a mechanism that couples catalysis to potential thiocyanate export .

How do mutations in the catalytic site affect YgaP function?

Several variants of YgaP have been studied to understand the role of key residues in its function:

These variants demonstrate the critical importance of Cys-63 in the catalytic mechanism of YgaP, confirming its role as the key residue in the formation of the enzyme-persulfide intermediate during sulfur transfer .

What evidence supports YgaP's role in thiocyanate export across the membrane?

Several lines of evidence support YgaP's potential role in thiocyanate export:

• Structural arrangement: YgaP is a membrane-embedded protein with a cytoplasmic catalytic domain and transmembrane helices, suggesting a role in coupling enzymatic activity with transport .

• Conformational changes: EPR analysis has demonstrated that the two second transmembrane helices of YgaP pack more tightly upon binding of the catalytic product SCN- . This structural rearrangement could create a pathway for thiocyanate export.

• Functional coupling: The catalytic reaction induces conformational changes not only within the rhodanese domain but also in the transmembrane helices of YgaP, suggesting communication between these regions during catalysis .

• Evolutionary context: As the only membrane-bound rhodanese domain protein in E. coli, YgaP's unique location suggests a specialized role that may involve both catalysis and transport .

These observations collectively provide insights into a potential mechanism for YgaP during its catalytic thiosulfate activity in vivo, suggesting it may function in both the detoxification of cyanide and the export of the resulting thiocyanate .

What are the most promising future research directions for understanding YgaP function?

Several promising research directions could advance our understanding of YgaP:

• In vivo studies: Investigating YgaP's role in E. coli cyanide resistance and thiocyanate export under physiological conditions.

• Protein dynamics: Further exploring the dynamic aspects of YgaP during catalysis, as dynamics appear to be involved in the mechanism of sulfurtransferase activity .

• Structure-function relationships: Detailed mapping of how structural changes in the transmembrane helices correlate with catalytic activity in the rhodanese domain.

• Interaction partners: Identifying potential protein partners that might interact with YgaP in the membrane environment.

• Comparative studies: Examining YgaP homologs in other bacterial species to understand evolutionary conservation and divergence of function.

What advanced imaging techniques beyond NMR and EPR could provide new insights into YgaP structure and function?

Advanced imaging techniques that could complement the existing NMR and EPR data include:

How might network analysis approaches contribute to understanding YgaP's role in cellular processes?

Network analysis approaches could provide valuable insights into YgaP's role within larger cellular contexts:

• Protein-protein interaction networks: Identifying interacting partners of YgaP could reveal its involvement in broader cellular processes beyond cyanide detoxification.

• Metabolic network analysis: Placing YgaP within the context of E. coli's metabolic networks could help understand its role in sulfur metabolism and detoxification pathways.

• Evolutionary network analysis: Comparing YgaP to homologous proteins across bacterial species could reveal evolutionary patterns and functional specialization.

• Systems biology approaches: Integrating transcriptomic, proteomic, and metabolomic data could provide a comprehensive view of YgaP's function under various environmental conditions.

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