Protein folding oligomeric proteins, concentration

Figure 3 The structural levels of proteins, exemplified by human insulin in the T6 form. (A) Primary structure residues 15-18 of human insulin B-chain, shown as sticks. (B) Secondary structure residues 8-20 of the B-chain form an a-helix, here depicted as a superposition of sticks, and a cartoon-representation. (C) Tertiary structure insulin A- and B-chains fold up to a monomer, which is assumed to be the active form, binding to the insulin receptor. Insulin can exist in different oligomeric forms, depending on formulation and protein concentration. (D) The Zn -stabilized hexamer form is shown. 2 Zn ions are bound per insulin hexamer (only one Zn2" "-ion is visible in this view). The hexamer is a trimer of dimmers. Figure based on pdb-file IMSO, produced in Pymol. Source Bente Vestergaard, Biostructural Research, Faculty of Pharmaceutical Sciences, University of Copenhagen.

Add a quantity of the crosslinker solution to the protein solution to provide a 1- to 10-fold molar excess of reagent over the concentration of protein. The use of lower molar ratios will limit the potential for oligomerization of proteins in solution. A series of reactions using different concentrations of crosslinker may have to be done to determine the optimal level to use for a particular application. 

The generic tendency of proteins to aggregate into nonfunctional, and sometimes cytotoxic, structures poses a universal problem for all types of cells. This problem is exacerbated by the high total concentrations of macromolecules found within most intracellular compartments, but it is solved by the actions of certain proteins that function as molecular chaperones. Different chaperones act by distinct mechanisms on both the folding of polypeptide chains and their subsequent assembly into oligomeric structures. Many chaperones, but not all, are also stress (or heat shock) proteins because the need for a chaperone function increases under stress conditions that cause proteins to unfold. 

If the protein does not oligomerize, then NMR studies can begin as long as a sample volume of 250 pL and concentration of 1 mM can be obtained. Good dispersion (separation) of the peaks in a one-dimensional spectrum indicates whether the protein is folded. The project can then proceed with the production of labeled protein and recording of 2D NMR spectra. 

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