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Research has been conducted on the manganese stabilizing protein. The functional and structural consequences of the protein's N-terminal elongation and truncation are discussed.

Research was conducted to identify specific amino acid(s) in a tripeptide at C-terminus of Manganese Stabilizing Protein (MSP) involved in the binding to photosystem II and restoring oxygen producing activity. Results from mutagenesis studies indicate that leucine 245 imparts stability to MSP and aids in the binding and oxygen production.

A study was conducted to examine the in vitro reconstitution of spinach photosystem II with recombinant manganese stabilizing protein supporting C-terminal deletions of amino acids. Oxygen evolution activity was determined polarographically at 25 degrees C while size-exclusion chromatography was carried out to determine the molecular masses of wild-type and mutant proteins. Results suggested that mutational effects on the binding and role of the protein may be correlated with tertiary structural changes.

Isotope editing and Fourier-transform infrared spectroscopy were used to elucidate the changes that take place in manganese stabilizing protein (MSP) upon functional reconstitution to photosystem II. The results showed consistency in the major conformational changes, with around 30 to 40% of the peptide structure exhibiting a change in secondary structure. The findings provided strong evidence that folding of MSP can be caused by the binding of MSP to the reaction center and that MSP can have a wide array of conformational states.

The binding of MSP with [V235A]MSP was analyzed to characterize the MSP binding sites and their role in the activity of the photosystem (PS) II. MSP prevented the binding of [V235A]MSP to PSII due to the direct competition of the proteins for two similar binding sites in PS II. Similar structural features were also required for the stable binding of MSP to the two binding sites in PS II. Furthermore, the binding of the wild-type protein to one PS II site strengthened the binding of the mutant protein in the other MSP binding site.

The substitution of Val235 with Ala induces an activation energy barrier for assembly of manganese-stabilizing protein (MSP) into photosystem II (PSII). Far-UV circular dichroism spectrometry studies reveal that low temperatures destabilize the V235A mutant MSP more than the wild type protein. Temperature however, affects the secondary structure of the mutant and the wild-type protein to a small extent. The mutation appears to suppress isomerization of one or more prolyl peptide bonds necessary for assembly of MSP into PSII.

An experiment to show that replacement of Arg151 or Arg161 yields proteins that have lower photosystem II (PSII) binding affinity, and are functionally deficient, even though about 2 mol of mutant MSP/mol PSII can be rebound to manganese-stabilizing protein (MSP) depleted PSII membranes. The results suggest that conserved positive charges on the manganese-stabilizing protein play a role in proper functional assembly of the protein into PSII, and consequently, in retention of Cl(super -) by the O(sub 2) - evolving complex.

The thermostability of the manganese stabilizing protein (MSP) of photosystem II was investigated by biochemical and spectroscopic methods. Samples of both native and recombinant spinach manganese stabilizing protein incubated at 90 deg C and cooled to 25 deg C were able to rebind to, and to reactivate, the O2-evolution activity of photosystem II membranes from which the native protein had been removed. Findings showed that MSP has the identifying features of a natively unfolded protein.

Manganese-stabilizing protein of photosystem II, an intrinsically disordered polypeptide, contains a high ratio of charged to hydrophobic amino acid residues. Argl51 and Argl61 are conserved in all known MSP sequences. To examine the role of these basic residues in MSP structure and function, three mutants of spinach MSP, R151G, R151D, and R161G, were produced. Here, we present evidence that replacement of Arg151 or Arg161 yields proteins that have lower PSII binding affinity, and are functionally deficient even though about 2 tool of mutant MSP/mol PSII can be rebound to MSP depleted PSII membranes. R161G reconstitutes O2 evolution activity to 40% of the control, while R151G and R151D reconstitute only 20% of the control activity. Spectroscopic and biochemical techniques fail to detect significant changes in solution structure. More extensive 02 evolution assays revealed that the Mn cluster is stable in samples reconstituted with each mutated MSP, and that all three Arg mutants have the same ability to retain Ca2+ as the wild-type protein. Activity assays exploring the effect of these mutations on retention of CI-, however, showed that the R151 G, R I 51 D, and R 161G MSPs are defective in Cl- binding to the OEC. The mutants have Cl- KM values that are about four (R161G) or six times (R151G and R151D) higher than the value for the wild-type protein. The results reported here suggest that conserved positive charges on the manganese-stabilizing protein play a role in proper functional assembly of the protein into PSII, and, consequently, in retention of Cl- by the O2-evolving complex. [ABSTRACT FROM AUTHOR]

The P22 tailspike adhesin is an elongated thermostable trimer resistant to protease digestion and to denaturation in sodium dodecyl sulfate. Monomeric, dimeric, and protrimeric folding and assembly intermediates lack this stability and are thermolabile. In the native trimer, three right-handed parallel β-helices (residues 143-540), pack side-by-side around the three-fold axis. After residue 540, these single chain β-helices terminate and residues 541-567 of the three polypeptide chains wrap around each other to form a three-stranded interdigitated β-helix. Three mutants located in this region - G546D, R563Q, and A575T - blocked formation of native tailspike trimers, and accumulated soluble forms of the mutant polypeptide chains within cells. The substitutions R563Q and A575T appeared to prevent stable association of partially folded monomers. G546D, in the interdigitated region of the chain, blocked tailspike folding at the transition from the partially-folded protrimer to the native trimer. The protrimer-like species accumulating in the G546D mutant melted out at 42°C and was trypsin and SDS sensitive. The G546D defect was not corrected by introduction of global suppressor mutations, which correct kinetic defects in β-helix folding. The simplest interpretation of these results is that the very high thermostability (Tm = 88°C), protease and detergent resistance of the native tailspike acquired in the protrimer-to-trimer transition, depends on the formation of the three-stranded interdigitated region. This interdigitated β-helix appears to function as a molecular clamp insuring thermostable subunit association in the native trimer. [ABSTRACT FROM AUTHOR]

The P22 tailspike adhesin is an elongated thermostable trimer resistant to protease digestion and to denaturation in sodium dodecyl sulfate. Monomeric, dimeric, and protrimeric folding and assembly intermediates lack this stability and are thermolabile. In the native trimer, three right-handed parallel beta-helices (residues 143-540), pack side-by-side around the three-fold axis. After residue 540, these single chain beta-helices terminate and residues 541-567 of the three polypeptide chains wrap around each other to form a three-stranded interdigitated beta-helix. Three mutants located in this region -- G546D, R563Q, and A575T -- blocked formation of native tailspike trimers, and accumulated soluble forms of the mutant polypeptide chains within cells. The substitutions R563Q and A575T appeared to prevent stable association of partially folded monomers. G546D, in the interdigitated region of the chain, blocked tailspike folding at the transition from the partially-folded protrimer to the native trimer. The protrimer-like species accumulating in the G546D mutant melted out at 42 degrees C and was trypsin and SDS sensitive. The G546D defect was not corrected by introduction of global suppressor mutations, which correct kinetic defects in beta-helix folding. The simplest interpretation of these results is that the very high thermostability (T(m) = 88 degrees C), protease and detergent resistance of the native tailspike acquired in the protrimer-to-trimer transition, depends on the formation of the three-stranded interdigitated region. This interdigitated beta-helix appears to function as a molecular clamp insuring thermostable subunit association in the native trimer.