The response of type three secretion system needle proteins MxiHDelta5, BsaLDelta5, and PrgIDelta5 to temperature and pH.
The type III secretion system (TTSS) is a specialized supramolecular injectisome composed of 25 or more proteins which form basal and extracellular domains and share gross architectural similarities with bacterial flagella. The extracellular component of the "needle complex" is primarily composed of a single monomeric subunit organized in a helical array surrounding a hollow pore and protrudes from the bacterial membrane. It is through this surface appendage that virulence factors are translocated to the host cell cytoplasm and thereby subvert normal host cell functions. We present here a comprehensive biophysical analysis of the dynamic conformational behavior of the truncated monomeric needle subunit proteins MxiH(Delta5) (Shigella flexneri), BsaL(Delta5) (Burkholderia pseudomallei), and PrgI(Delta5) (Salmonella typhimurium) as well as their thermal stability over a pH range of 3-8. Circular dichroism spectroscopy indicates the secondary structure is largely alpha helical in all three proteins, and surprisingly thermally labile with transition midpoints in the range of 35-50 degrees C over the pH range of 3-8. Additionally, at the concentrations examined, the very broad thermal transitions were >90% reversible. Second derivative UV absorbance spectroscopy data indicates some disruption of the protein's tertiary structure occurs at temperatures in the range of 29-46 degrees C. The difference in the pH of maximal stability for each of the proteins and the variation for each protein with respect to both secondary and tertiary structural elements is striking. It appears, that at physiological temperatures all three proteins experience intermediate non-native molten globule like states in which they display significant secondary structure in the absence of extensive tertiary interactions. Because of the size difference between the inner pore of the needle and the fully folded needle proteins, it seems clear that the needle subunits must be secreted in a partially or completely unfolded state to reach the distal tip of the needle for assembly. It is proposed that the formation of these intermediate states in the physiological temperature range may play a role in passage through the pore and needle assembly.
Barrett
Brooke S
BS
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047, USA.
Picking
Wendy L
WL
Picking
William D
WD
Middaugh
C Russell
CR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D016957:Burkholderia pseudomallei / Q000737:chemistry*; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D012486:Salmonella typhimurium / Q000737:chemistry*; D016415:Sequence Alignment; D012763:Shigella flexneri / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013451:Sulfonic Acids / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D001426:Bacterial Proteins; D050505:Mutant Proteins; C413572:MxiH protein, Shigella; D013451:Sulfonic Acids; D014364:Tryptophan