Plimstex Curves Under Different HoloCaM Concentrations

As demonstrated earlier (Fig. 11.2), PLIMSTEX curves are sensitive to the total protein concentration and do not yield reliable K values when the protein is titrated at high concentrations (@100 times the 1/K or Kd). Nevertheless, when the concentration is too high, "sharp-break" curves (curve B in Fig. 11.2, curve b in Fig. 11.7) are obtained and can be used for stoichiometry determination. We found the binding stoichiometry for holo-CaM: melittin binding to be 1:1 by titrating a relatively high concentration of holo-CaM (15 |M) with melittin (Fig. 11.7, curve a). Interestingly, we found that the binding of mastoparan, which is a 14-amino-acid residue peptide from the wasp and is approximately half the size of melittin, is accompanied by a greater loss of solvent accessibility for CaM than that caused by binding of melittin (Fig. 11.7, curve b), ruling out a direct block of the surface amides, and indicating significant conformational change (additional H-bonding) with the binding. The PLIMSTEX result is in accord with the proposed structure of the holo-CaM:melittin complex [65] for which the holo-CaM changes from an open dumbbell shape to a closed globular shape with both domains interacting with the peptide. The conformational change induced by mastoparan binding may cause the small peptide to be surrounded by the two domains of CaM, whereas this full interaction may not be possible for the longer peptide melittin. These two examples show the opportunity for PLIM-STEX to suggest conformational changes associated with protein-ligand binding.

Owing to the high sensitivity of mass spectrometers and the chromatographic concentrating procedure in our protocol, we are able to measure a wide range of protein concentrations in PLIMSTEX by simply adjusting the injection for MS analysis. Small quantities (high picomolar) and low concentration (nanomolar) of proteins are sufficient for mass measurement for each point in a titration. To determine the binding affinity between the holo-CaM and melittin, we had to lower the protein concentration from 15.0 mM to 0.15 mM and redo the titration. The resulting K and DD1 for holo-CaM are (5.4 + 0.9) x 107 M_1 and 29.3 + 0.3 (Table 11.1), respectively, indicating a strong interaction occurs in the binding of melittin and holo-CaM accompanied by protection of @29 backbone amide hydrogens. Although there is a wide range of binding constants [(0.93-33.0) x 107 M_1] in the literature [58, 59], the latter constant, which is commonly cited, was determined by using an affinity column to separate free [3H] mono-acetyl-melittin from CaM-bound melittin and quantify it by liquid scintillation counting [58]. If the high affinity were correct, then the most appropriate protein concentration for the titration would not be 150 nM, but 3 nM, a concentration that challenges current MS.

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Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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