Blood -- Structural and functional effects of hereditary hemolytic anemia-associated point mutations in the alpha spectrin tetramer site

Blood, Vol. 111, Issue 12, 5712-5720, June 15, 2008

Structural and functional effects of hereditary hemolytic anemia-associated point mutations in the alpha spectrin tetramer site
Blood Gaetani et al. 111: 5712

Supplemental materials for: Gaetani et al

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Figure S1. CD spectra of wild type and mutant α0-1 spectrin peptides (JPG, 74.4 KB) -
Samples were analyzed in phosphate buffered saline, pH 7.4 at room temperature. The spectrum of the wild type 0-1 recombinant is repeated in each panel for comparison.
Figure S2. Sedimentation equilibrium analysis of α0-1 spectrin peptides (JPG, 87.1 KB) -
Representative data from sedimentation equilibrium analyses of recombinant proteins in 20 mM Tris, 130 mM NaCl, 1 mM EDTA, 0.1 mM PMSF and 1 mM TCEP, pH 7.4, at 28,000 rpm and 4°C are shown. (A) 0-1 wild type recombinant protein with three initial loading concentrations of 1.5, 0.75 and 0.375 mg/ml. (B) the I24S 0-1 recombinant protein is shown as a representative case (all other mutants produced comparable results) with three initial loading concentrations of 1.0, 0. 5 and 0.275 mg/ml. Lower panels – Raw data are shown using circles and global fits for a monomer species are indicated by lines, although these lines are obscured by the data symbols. Upper panels – The residuals of the fitted curves to the data points are represented for each concentration from the highest to the lowest, from top to bottom respectively.
Figure S3. Representative tetramer site gel filtration assays (JPG, 55.7 KB) -
Preliminary time course experiments were conducted using wild type 0-1 and 16-17, which verified equilibrium was reached within 1 hr at 37°C. For subsequent experiments 500 pmoles 0-1 (wild type or mutant) and 500 pmoles 16-17 were mixed, incubated 1 hr at 37°C, and immediately injected onto a TSKgel Super SW3000 (4.6 mm I.D. × 30 cm length, Tosoh Bioscience) column equilibrated with 10 mM sodium phosphate, 130 mM NaCl, 1 mM EDTA, 0.15 mM PMSF, 1 mM -ME, pH 7.4. Peaks were eluted at 0.1 ml/min with detection of absorbance at 280 nm. The 0-1/16-17 protein complexes eluted at approximately 35 min, while unbound recombinant proteins eluted at approximately 40 min. Top panel, binding assay of 0-1 K48R + 16-17 ( ^____ ), 0-1 K48R only (^...), 16-17 only ( --- ); binding affinity in this case is similar to wild type (data not shown). Center panel, binding assay of 0-1 G46V + 16-17 ( ^____ ), 0-1 G46V alone ( ^... ), 16-17 alone ( --- ); binding affinity in this case is substantially weaker than wild type as indicated by reduced amount of complex and increased amount of individual components. Bottom panel, binding assay of 0-1 R28H +16-17 ( ^____ ), 0-1 R28H alone ( ^... ), 16-17 alone ( --- ); there was no detectable binding in this case as no complex was detected.