The denatured state of a protein contains important information about the determinants of the folding process. By combining site-directed spin-labeling NMR experiments and restrained computer simulations, we have determined ensembles of conformations that represent the denatured state of the bovine acyl-coenzyme A binding protein (ACBP) at three different concentrations of guanidine hydrochloride. As the experimentally determined distance information corresponds to weighted averages over a broad ensemble of structures, we applied the experimental restraints to a system of noninteracting replicas of the protein by using a Monte Carlo sampling scheme. This procedure permits us to sample ensembles of conformations that are compatible with the experimental data and thus to obtain information regarding the distribution of structures in the denatured state. Our results show that the denatured state of ACBP is highly heterogeneous. The high sensitivity of the computational method that we present, however, enabled us to identify long-range interactions between two regions, located near the N- and C-termini, that include both native and non-native elements. The preferential formation of these contacts suggests that the sequence-dependent patterns of helical propensity and hydrophobicity are important determinants of the structure in the denatured state of ACBP.

No abstract

No abstract

Euphauserase is a brachyurin type digestive enzyme isolated from Antarctic krill. The brachyurins belong to clan SA of the S1 family of serine endopeptidases. In this study, we demonstrate that the precursor form of recombinant euphauserase, termed pro-r-euphauserase, can be successfully expressed in Pichia pastoris. The presence of most of the 51-residue euphauserase propeptide is essential during expression, under the growth conditions of Pichia. The propeptide may be required either for correct folding or processing of the enzyme. Cod trypsin generates a fully active r-euphauserase from its precursor, which appears to be identical to the native enzyme. The mature r-euphauserase sequence contains 250 amino-acid residues including a 13-residue activation peptide, which seems to be attached to the molecule by a disulfide bond. Euphauserase shares an average sequence identity of 62% with its type I brachyurin analogue, crab collagenase I. However, the identity between these two sequences is much higher in the regions shown to be important for the broad substrate specificity and collagen binding of crab collagenase I. The type I brachyurins share only 30±40% identities with the type II brachyurins and trypsins. The low isoelectric point of euphauserase, with a calculated pI value of 3.9, is typical for the type I brachyurins. [14]. These enzymes are presently classified into three types, termed Ia, Ib and II, based on their substrate specificity [3]. The distinctive characteristic of many brachyurins, in particular those of type I, is their ability to cleave collagen [9,10,14,15]. In addition, the type I brachyurins show chymotrypsin-like, trypsin-like and elastaselike activities towards synthetic substrates. The substrate specificity of the broadly specific type I brachyurins, such as crab collagenase I and euphauserase, differ from those of type II, which are strictly trypsin like [3].Fiddler crab collagenase I was the first serine protease shown to possess collagenolytic activity and thus serves as the paradigm for the brachyurins [3,15]. Its crystal structure in complex with the serine protease specific inhibitor ecotin revealed some of the structural features required for its broad substrate specificity and ability to cleave collagen [16,17].Euphauserase shares 62% amino sequence identity with crab collagenase I. Moreover, most of the regions in crab collagenase I shown to be important for its broad substrate specificity and collagen binding are highly conserved in the euhauserase sequence. This is reflected in the enzymatic characteristics of euphauserase such as collagenolytic activity as well as broad substrate specificity [18].All serine peptidases are synthesized as preproenzymes composed of an N-terminal signal sequence and a propeptide of variable length requiring proteolytic cleavage for enzyme activation. The role of prosequences in vertebrate proteins is unclear. In some cases these have been shown to be essential for correct folding of bacterial [19,20] and yeast [21] enzyme precursors that are ei...