Biomedical subjects

Yellamaraju Sreelakshmi

Publications and source records attributed to Yellamaraju Sreelakshmi.

4 recordsLinked to original sources

PubMed2006

The interaction between alphaA- and alphaB-crystallin is sequence-specific.

PURPOSE: We have previously shown that residue 42-57 (TSLSPFYLRPPSFLRA; recognition sequence 1 or RS-1) and residue 60-71 (WFDTGLSEMRLE; recognition sequence 2 or RS-2) in alphaB-crystallin play a role in oligomerization and subunit interaction with alphaA-crystallin. When we created multiple mutations in alphaB-crystallin in RS-1 and RS-2 at S53(T), F54(G), L55(G), W60(R), and F61(N), we found that these mutations destabilized the protein, and the protein precipitated. When the individual mutations were created at F54, W60, and F61 in alphaB-crystallin, protein stability was not affected, but the mutations had an effect on oligomerization and subunit interaction with alphaA-crystallin. To find out whether the sequence specificity of these residues is important for the overall function of alphaB-crystallin, we inverted the 54-60 sequence such that 54FLRAPSW60 became 54WSPARLF60 using site-directed mutagenesis. We studied the effect of inversion on oligomerization and subunit interaction with alphaA-crystallin. METHODS: Mutations were introduced using site-directed mutagenesis and the mutant protein, expressed in Escherichia coli BL21(DE3)pLysS cells, was purified by ion-exchange and gel filtration chromatography. The mutation was confirmed by mass spectrometry. The structure and hydrophobicity were analyzed by spectroscopic methods. The chaperone-like activities of wild-type and mutant proteins were compared using alcohol dehydrogenase and citrate synthase. Subunit exchange between alphaA- and alphaB-crystallin was monitored by fluorescence resonance energy transfer (FRET). For this purpose, purified alphaB- and alphaBinvert-crystallin were labeled with Alexa fluor 350 whereas Alexa fluor 488 was used to label alphaA-crystallin. RESULTS: The inversion of residues 54-60 led to homooligomers that were 38% smaller in size than their wild-type counterparts. The inversion also reduced the tryptophan fluorescence intensity by 50%, as compared to that of wild-type alphaB-crystallin. This suggests that Trp54 is less exposed than Trp60. Inversion of residues did not affect the total hydrophobicity in alphaB-crystallin. Secondary structural analysis revealed a slight increase in the alpha-helical content of alphaBinvert-crystallin protein as compared to wild-type alphaB-crystallin. Except for an increase in the ellipticity of the alphaBinvert-crystallin mutant, no change was observed in the tertiary structure, as compared with that of wild-type alphaB-crystallin. Chaperone-like function was similar in the alphaBinvert-crystallin mutant and wild-type alphaB-crystallin. The inversion of residues decreased the subunit exchange rate with alphaA-crystallin by two fold. CONCLUSIONS: This study establishes for the first time that proper orientation of residues contributing to RS-1 and RS-2 sites in alphaB-crystallin is important for homooligomerization and optimal subunit interaction with alphaA-crystallin.

Amino Acid Sequence↗

PubMed2005

Recognition sequence 2 (residues 60-71) plays a role in oligomerization and exchange dynamics of alphaB-crystallin.

Previously, using the peptide scan method, we have determined that residues 42-57 and 60-71 in alphaB-crystallin (TSLSPFYLRPPSFLRA, named recognition sequence 1 or RS-1, and WFDTGLSEMRLE, named recognition sequence 2 or RS-2) are involved in interaction with alphaA-crystallin. To understand the significance of the RS-2 region in interactions between alphaA- and alphaB-crystallins, W60R, F61N, and S66G mutants of alphaB-crystallin were made and tested for their ability to interact with alphaA-crystallin. W60R and S66G mutations increased the oligomeric size of alphaB-crystallin by 1.6- and 2.7-fold respectively, whereas the F61N mutation had no effect. The tryptophan fluorescence intensity of alphaBS66G was 1.5-fold higher than that for the wild type. The intrinsic fluorescence of alphaBF61N was marginally lower than that of alphaB, whereas the fluorescence intensity of alphaBW60R decreased by 40% compared with that of alphaB. The relative availability of hydrophobic sites in the mutants was in the following order: alphaBS66G >> alphaB = alphaBF61N = alphaBW60R. The far-UV CD profiles for the wild type and alphaB-crystallin mutants indicated no significant changes in their secondary structures, except for alphaBS66G, which showed an increase in alpha-helical content. The near-UV CD profiles of alphaBW60R and alphaBF61N were nearly similar to that of wild type alphaB. On the other hand, alphaBS66G beyond 270 nm exhibited a signature completely different from that of wild type alphaB. Mutations did not alter the chaperone-like activity of these proteins. The W60R mutation did not affect the rate of subunit exchange between alphaB- and alphaA-crystallins. On the other hand, the S66G mutation increased the subunit exchange rate by 100%, whereas the F61N mutation decreased the rate of subunit exchange between alphaB- and alphaA-crystallins by 36%. Our results establish the importance of residues 60-71 in oligomerization of alphaB-crystallin and subunit interaction between alphaB- and alphaA-crystallins.

Circular Dichroism↗

PubMed2004

AlphaA-crystallin interacting regions in the small heat shock protein, alphaB-crystallin.

Amino acid sequences of alphaB-crystallin, involved in interaction with alphaA-crystallin, were determined by using peptide scans. Positionally addressable 20-mer overlapping peptides, representing the entire sequence of alphaB-crystallin, were synthesized on a PVDF membrane. The membrane was blocked with albumin and incubated with purified alphaA-crystallin. Probing the membrane with alphaA-crystallin-specific antibodies revealed residues 42-57, 60-71, and 88-123 in alphaB-crystallin to interact with alphaA-crystallin. Residues 42-57 and 60-71 interacted more strongly with alphaA-crystallin than the 88-123 sequence of alphaB-crystallin. Binding of one of the alphaB peptides (42-57) to alphaA-crystallin was also confirmed by gel filtration studies and HPLC analysis. The alphaB-crystallin sequences involved in interaction with alphaA-crystallin were distinct from the chaperone sites reported earlier as binding of the alphaB sequence from residues 42-57 does not alter the chaperone-like function of alphaA-crystallin. To identify the critical residues involved in interaction with alphaA-crystallin, R50G and P51A mutants of alphaB-crystallin were made and tested for their ability to interact with alphaA-crystallin. The oligomeric size and hydrophobicity of the mutants were similar. Circular dichroism studies showed that the P51A mutation increased the alpha-helical content of the protein. While the alphaBR50G mutant showed chaperone-like activity similar to wild-type alphaB, alphaBP51A showed reduced chaperone function. Fluorescence resonance energy transfer studies showed that the P51A mutation decreased the rate of subunit exchange with alphaA by 63%, whereas the R50G mutation reduced the exchange rate by 23%. Similar to wild-type alphaB, alphaB-crystallin peptide (42-57) effectively competed with alphaBP51A and alphaBR50G for interaction with alphaA. Thus, our studies showed that the alphaB-crystallin sequence (42-57) is one of the interacting regions in alphaB and alphaA oligomer formation.

Amino Acid Sequence↗

PubMed2003

The polycotyledon mutant of tomato shows enhanced polar auxin transport.

The polycotyledon mutant of tomato (Lycopersicon esculentum L. cv Ailsa Craig) showed altered development during embryogenesis and during vegetative and reproductive phases. The phenotype was pleiotropic and included the formation of extra cotyledons, changes in leaf shape, increased number of flowers (indeterminacy) with abnormal floral organs, the formation of epiphyllous structures, and altered gravitropism. The earliest defects were observed at the transition from the globular to the heart stage of embryogenesis with the formation of multiple cotyledons. Epidermal cells in the mutant embryo were smaller and less expanded compared with wild type. Examination of polar auxin transport (PAT) showed a striking enhancement in the case of the mutant. Increase in PAT did not appear to be caused by a decrease in flavonoids because the mutant had normal flavonoid levels. Application of 2,3,5-triiodobenzoic acid, an inhibitor of polar transport of auxin, rescued postgermination phenotypes of young seedlings. Our analysis reveals a level of control that negatively regulates PAT in tomato and its contribution to plant development and organogenesis.

Biological Transport↗