| Protein folding remains one of the last mysteries remaining in the field of molecular biology. Understanding the process of protein folding will likely be critical to design of novel proteins in the future. Two-state proteins only exist in either the unfolded or native states, so examination of the process of folding is targeted at the high-energy transition state barrier. The transition state of common-type acyl phosphatase (ctAcP) is characterized using psi-analysis which identifies chain-chain contacts using engineered bi-histidine metal ion binding sites located throughout the protein. As with ubiquitin 1, the other globular protein extensively characterized using psi-analysis, the transition state of ctAcP has very native-like topology. Using multiple-metal folding analysis, it was determined that very little pathway heterogeneity exists at the rate-limiting step, indicating the transition state ensemble of ctAcP has single consensus structure with a minor amount of optional elements. Using hydrogen exchange results, models of several transition states were generated and found to be very native-like in topology. Based on these results and previous correlations between topology and folding rate, we believe proteins who obey a linear relationship between topological complexity and folding activation energy are likely to retain ∼80% of native topology in the transition state. This result experimentally confirms that arrangement of the polypeptide chain into a native-like configuration is the rate limiting step in protein folding. |
