Onconase (ONC) from Rana pipiens is the smallest member of the ribonuclease A (RNase A) superfamily. Despite a tertiary structure similar to RNase A, ONC is distinguished by an extremely high thermodynamic stability. In the present paper we have probed the significance of three structural regions, which exhibit structural peculiarities in comparison to RNase A, for the stability of ONC to temperature and guanidine hydrochloride induced denaturation: (i) the N-terminal pyroglutamate residue, (ii) the hydrophobic cluster between helix I and the first beta-sheet, and (iii) the C-terminal disulfide bond. For this purpose, the enzyme variants
Aging population and longer life expectancy are the main reasons for an increasing number of patients with wound problems. Although the interest in wound care increases continuously, wound management still remains a challenge mainly due to the higher occurrence of chronic wounds, which require intensive care and constant monitoring. Here, we demonstrate a fluorescent sensing system to monitor the wound status and to distinguish between an autonomously healing and a chronic wound at an early stage. The system allows monitoring two of the most relevant fluctuating wound parameters during the healing process which are pH and glucose concentration. A fluorescent pH indicator dye, carboxynaphthofluorescein, and a metabolite-sensing enzymatic system, based on glucose oxidase and horseradish peroxidase, were immobilized on a biocompatible polysaccharide matrix to develop a functional hydrogel coating for wound monitoring. The changes in metabolite and enzyme concentration in artificial wound extract were converted into a fluorescent signal.
A promising approach to unravel the relationship between sequence information, tertiary structure, and folding mechanism of proteins is the analysis of the folding behavior of proteins with low sequence identity but comparable tertiary structures. Ribonuclease A (RNase A) and its homologues, forming the RNase A superfamily, provide an excellent model system for respective studies. RNase A has been used extensively as a model protein for folding studies. However, little is known about the folding of homologous RNases. Here, we analyze the folding pathway of onconase, a homologous protein from the Northern leopard frog with great potential as a tumor therapeutic, by high-resolution techniques. Although onconase and RNase A significantly differ in the primary structure (28% sequence identity) and in thermodynamic stability (DeltaDeltaG = 20 kJ mol(-1)), both enzymes possess very similar tertiary structures. The present folding studies on onconase by rapid mixing techniques in combination with fluorescence and NMR spectroscopy allow the structural assignment of the three kinetic phases observed in stopped-flow fluorescence spectroscopy. After a slow peptidyl-prolyl cis-to-trans isomerization reaction in the unfolded state, ONC folds via an on-pathway intermediate to the native state. By quenched-flow hydrogen/deuterium exchange experiments coupled with 2D NMR spectroscopy, 31 amino acid residues were identified to be involved in the structure formation of the intermediate. Twelve of these residues are identical in the RNase A sequence, which is a significantly higher percentage (39%) than the overall 28% sequence identity. Moreover, the structure of this intermediate closely resembles two of the intermediates that occur early during the refolding of RNase A. Obviously, in spite of considerable differences in their amino acid sequence the initial folding events of both proteins are comparable, guided by a limited number of conserved residues.
Though lacking a well-defined three-dimensional structure, intrinsically unstructured proteins are ubiquitous in nature. These molecules play crucial roles in many cellular processes, especially signaling and regulation. Surprisingly, even enzyme catalysis can tolerate substantial disorder. This observation contravenes conventional wisdom but is relevant to an understanding of how protein dynamics modulates enzyme function. This chapter reviews properties and characteristics of disordered proteins, emphasizing examples of enzymes that lack defined structures, and considers implications of structural disorder for catalytic efficiency and evolution.
Enzymes have evolved to increase chemical reaction rates, some by factors exceeding the trillions, thus enabling the remarkable success of life on Earth. A typical enzymatic process includes substrate binding, a chemical step involving covalent bond rearrangements, and product release. A distinct energy threshold must be overcome for each of these steps to proceed. Past studies of enzyme evolution have focused on how the overall catalytic process or specific steps such as binding respond to selective pressures, but researchers have not deliberately monitored the evolution of the chemical step per se until now. To study the chemical step, we measured the temperature dependence of intrinsic kinetic isotope effects of dihydrofolate reductase from primitive to evolved variants. We found a progressive decrease in the temperature dependence of intrinsic kinetic isotope effects with evolution, indicating gradual narrowing of the thermally averaged donor–acceptor distance for hydride transfer in step with an increased catalytic efficiency. The important role played by residues that are remote from the active site in optimizing the chemical step of this complex, multistep enzymatic pathway is particularly notable.
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