Productive folding pathways

Have we exhausted catalases as a source of information about protein structure and the catalatic mechanisms The answer is clearly no. With each structure reported comes new information, often including structural modifications seemingly unique to catalases and with roles that remain to be explained. Despite a deeply buried active site, catalases exhibit one of the fastest turnover rates determined. This presents the as yet unanswered question of how substrate can access the active site while products are simultaneously exhausted with a potential turnover rate of up to 10 per second. The complex folding pathway that produces the intricate interwoven arrangement of subunits also remains to be fully clarified. 

Figure 18.7 Energy profiles for compact states in the folding reaction. Left The fully unfolded state collapses to a genuine intermediate that rearranges to form products. Right. The compact state C is off the pathway and has to unfold for productive folding to occur.

Structure analysis of several proteases involved in blood coagulation and fibrinolysis reveals a diverse, sometimes repetitive, assembly of discrete protein modules (Fig. 9.4) [56]. While these modules represent independent structural units with individual folding pathways, their concerted action contributes to function and specificity in the final protein product. On the genetic level, these individual modules are encoded in separate exons. Over the course of modular protein evolution, new genes are created by duplication, deletion, and rearrangement of these exons. Mechanistically, the exon shuffling actually takes place in the intervening intron sequences (intronic recombination - for further details see [10]). 

Figure 3. Intracellular folding pathway of P22 tailspike proteins. The newly synthesized wild type or mutant polypeptide chains at 30°C first fold into partially folded monomeric intermediates. These species fold and associate to form a protrimer intermediate. Further folding results in a thermostable native tailspike. At 40°C, the folding is inhibited and tsf mutants act by blocking an early step in chain folding, prior to association. However, if infected cells are shifted to 30 C, the mutant chains continue through the productive pathway.

The first approach is termed folding pathway [42, 43] and uses a common set of reaction conditions to transform a range of substrates into products with distinct and diverse molecular skeletons (Figure 27.1a). The substrates are encoded to fold into the alternative scaffolds through strategically embodied functionalities, known as a-elements. Each c-element thus dictates the formation of diverse molecular framework. 

The scope of the approach is extremely broad and, indeed, some folding pathways e.g. Scheme 1.4 Section 1.2.3.1) and branching pathways e.g. Scheme 1.5 Section 1.2.3.2) can be considered to exemplify the build-couple-pair strategy. For example, the four component Petasis reaction illustrated in Scheme 1.4 allowed simple building blocks to be combined complementary cyclisation reactions were then use to pair functional groups to yield a diverse range of product scalfolds. 

The above work by Oguri and Schreiber can be considered to be an example of the build/ couple/pair strategy as the initial building blocks A, B, and C must first be built, then coupled to the piperidinone template, and finally the reactive functionality paired inlramolecularly to yield the products. Another example of the build/couple/pair strategy incorporating a folding pathway can be found in the work of Mitchell and Shaw (Scheme 4.6). ... 

Yet the unfolded 1-D chain does fold into a functionally active unit, which maintains a reasonably well defined structure, within certain limits. It does so in a very short time, far shorter than any estimated time that would have been required by the random selection of a folding pathway. This means that there exists not only a thermodynamic force leading from the unfolded to the folded state, but there must be some dynamical forces, at least in part of the folding pathway, that direct the protein to move toward the final product. 

Having taken care of the hard and the soft interactions, the only interactions left are the solute-solvent HBing capacity of the OH of serine as well as the C=0 and NH of the backbone. Even in this simple polypeptide we have to consider a multitude of very large effects which will determine both the folding pathway and the final product. Some of these are ... 

---