Protein folding time constraints

Time constraints. The time to synthesize proteins routinely ranges from a few seconds to no more than a few minutes. According to one prominent protein-folding model, a newly made polypeptide tries out all possible conformations until the most stable one is achieved. Calculations of the time required for every bond in a small protein molecule to rotate until the final biologically active form is achieved indicate that an astronomical number of years would be required. Even when only a smaller number of possible bond rotations are considered, the time required for folding is still measured in years. Therefore most researchers have concluded that protein folding is not a random process based solely on primary sequence. 

The relationship between NMR chemical shifts and the secondary structure of a protein has been well established (19,20,21). The C and carbonyl carbons experience an upfield shift in extended structures, such as a P-strand, and a downfield shift in helical structures. Both the Cp and the Ha proton chemical shifts exhibit the opposite correlation. These shifts have proven to be sufficiently consistent to permit the prediction of secondary structural elements for a number of proteins (1,19,20). Knowledge of the secondary structure of a protein can be useful in identifying spin-diffusion effects during the analysis of 4D N/ N-separated NOES Y data collected with long mixing times as described below. The secondary structure can also be used as a constraint in the calculation of protein global folds. 

An alternative method of analysis of NOESY data, which is usually sufficient for resolved peaks with a digital resolution much greater than the intrinsic line width and coupling constants, is to measure the maximum peak amplitude or to count the number of contours. NOESY cross peaks can then be classified as strong, medium or weak and can be translated into upper distance restraints of around 2.5, 3.5 and 5.0 A respectively. The lower distance constraint is usually the sum of the van der Waals radii (1.8 A for protons). This simple approach is reasonably insensitive to the effects of spin diffusion or non-uniform correlation times and can usually lead to definition of the global fold of the protein, provided a sufficiently large number of NOEs have been identified. 

As was the case with the solution structural studies on the mammalian proteins [154,219-222], there were no dipolar crmstraints between the two metal domains to permit their respective orientation. This was a limitation when working with MTs isolated from natural source at the time, which today could be overcome by the expression of recombinant MT with isotopic labeling and the use of residual dipolar coupling methods [227]. Another advancement in the 3D structural studies on this protein has arisen from the increased sensitivity of newer, higher field NMR instruments. NMR studies on MT at 800 MHz now provide a sufficient number of H- H NOE dipolar constraints to permit the calculation of the tertiary fold of the protein backbone in MT without the need for Cd substitution and the... 

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