BackgroundDisulfide engineering is an important biotechnological tool that has advanced a wide range of research. The introduction of novel disulfide bonds into proteins has been used extensively to improve protein stability, modify functional characteristics, and to assist in the study of protein dynamics. Successful use of this technology is greatly enhanced by software that can predict pairs of residues that will likely form a disulfide bond if mutated to cysteines.ResultsWe had previously developed and distributed software for this purpose: Disulfide by Design (DbD). The original DbD program has been widely used; however, it has a number of limitations including a Windows platform dependency. Here, we introduce Disulfide by Design 2.0 (DbD2), a web-based, platform-independent application that significantly extends functionality, visualization, and analysis capabilities beyond the original program. Among the enhancements to the software is the ability to analyze the B-factor of protein regions involved in predicted disulfide bonds. Importantly, this feature facilitates the identification of potential disulfides that are not only likely to form but are also expected to provide improved thermal stability to the protein.ConclusionsDbD2 provides platform-independent access and significantly extends the original functionality of DbD. A web server hosting DbD2 is provided at http://cptweb.cpt.wayne.edu/DbD2/.

Single molecules of alkaline phosphatase are captured in a capillary filled with a fluorogenic substrate. During incubation, each enzyme molecule creates a pool of fluorescent product. After incubation, the product is swept through a high-sensitivity laser-induced fluorescence detector; the area of the peak provides a precise measure of the activity of each molecule. Three studies are performed on captured enzyme molecules. In the first study, replicate incubations are performed on the same molecule at constant temperature; the amount of product increases linearly with incubation time. Single enzyme molecules show a range of activity; the most active molecules have over a 10-fold higher activity than the least active molecules. In the second study, replicate incubations are performed on the same molecule at successively higher temperatures. The activation energy of the reaction catalyzed by a single molecule is determined with high precision. Single enzyme molecules show a range of activation energy; microheterogeneity extends to thermodynamic properties of catalysis. The average activation energy is within experimental error of the activation energy obtained from analysis of a bulk sample. These results are consistent with the first postulate of statistical thermodynamics:  a thermodynamic property obtained from the time average of an individual molecule is identical to that produced by an ensemble average over a large number of molecules. In the third study, the activity of single enzyme molecules is measured after partial heat denaturation. The number of active molecules decreases in proportion to the extent of denaturation. However, the activity of the surviving molecules is experimentally indistinguishable from the activity of control enzyme. Thermal denaturation of alkaline phosphatase is a catastrophic process, wherein the molecule undergoes irreversible conversion to an inactive form.

a b s t r a c tImproving the stability of proteins is an important goal in many biomedical and industrial applications. A logical approach is to emulate stabilizing molecular interactions found in nature. Disulfide bonds are covalent interactions that provide substantial stability to many proteins and conform to well-defined geometric conformations, thus making them appealing candidates in protein engineering efforts. Disulfide engineering is the directed design of novel disulfide bonds into target proteins. This important biotechnological tool has achieved considerable success in a wide range of applications, yet the rules that govern the stabilizing effects of disulfide bonds are not fully characterized. Contrary to expectations, many designed disulfide bonds have resulted in decreased stability of the modified protein. We review progress in disulfide engineering, with an emphasis on the issue of stability and computational methods that facilitate engineering efforts. Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Disulfide bonds in proteinsA protein disulfide bond is a covalent link between the sulfur atoms of the thiol groups (-SH) in two cysteine residues. The disulfide (also called an SS-bond, disulfide bridge, or crosslink) is formed upon oxidation of the two thiols, thus linking the two cysteines and their respective main peptide chains by the covalent disulfide bond. Conversely, a disulfide bond can be disrupted by a reductive reaction (e.g. using dithiothreitol).Disulfide bonds are found predominantly in secreted extracellular proteins. The redox environment within the cytosol preserves cysteine sulfhydryls in a reduced state. Disulfide bonds rapidly form outside of the cell in the presence of oxygen.Most disulfide bonds in proteins secreted from eukaryotic cells are formed in the endoplasmic reticulum, which offers an oxidizing environment as well as chaperones and disulfide isomerases to ensure correct protein folding and disulfide connectivity [1].In proteins, disulfide bonds are a configuration of six atoms,a , linking two cysteine residues. The seminal work of Janet Thornton in 1981 characterized the features and bond geometry of disulfides by analyzing the atomic coordinates of 55 disulfide bonds that existed in protein structures available at the time [2]. Nearly twenty years later, disulfide bond features were further detailed by Petersen et al. using 351 disulfide bonds in 131 non-homologous protein structures [3]. These analyses revealed the distribution of bond angles and distances found in naturally occurring disulfides, and this work has provided the basis of most models for disulfide engineering. Two important bond angles are the Ca-Cb-Sc and Cb-Sc-Sc (Fig. 1) 69]). These values are slightly different from the often-cited ±90°. This torsion angle is critical to the stability of a disulfide bond, and deviations from optimal values can produce an energy strain by several kcal/mol [4,5]. The v 1 torsion angle, defined by the N-Ca-Cb-Sc bo...

Glowing marks: A new class of protein stains, the pyrylium dyes, undergo a strong color change (typically from blue to red, see picture) on covalently binding to proteins. While the free stains are almost nonfluorescent, the protein‐conjugated forms are highly fluorescent. The dyes do not alter the charge of a protein, and thus do not change its electrophoretic properties. The stains also can be used in quantitative protein assays.

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Fluorescent dyes are often used to label proteins before analysis by capillary electrophoresis. Fluorescent labeling produces spectacular improvements in sensitivity compared with UV absorbance detection of the native protein. However, labeling of the protein can lead to significant band broadening. This band broadening is interpreted as a result of multiple labeling of the protein, wherein one or more fluorescent molecules are bound to the protein. The heterogeneous reaction products, which are presumed to have different mobilities, generate a broad peak during electrophoresis. There has been little direct evidence for multiple labeling as the cause of band broadening of proteins. In this paper, we perform electrophoresis on native green fluorescence protein, along with the reaction products produced by fluorescence labeling. For short incubations, a series of regularly spaced components are resolved by free-zone electrophoresis; upon longer incubation, the product peaks merge together, forming a broad envelope.

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Py-1 and Py-6 are novel amino-reactive fluorescent reagents. The names given to them reflect that they consist of a pyrylium group attached to small aromatic moieties. Upon reaction with a primary amine there is a large spectral shift in the reagent, rendering them effectively fluorogenic. In this study, these reagents were used to label a test protein, (human serum albumin), and the sample was analyzed by capillary electrophoresis and laser-induced fluorescence detection. Detection limits after a 60 min labeling reaction at 22 degrees C (Py-1) and 50 degrees C (Py-6) were 6.5 ng/mL (98 pM) for Py-1 and 1.2 ng/mL (18 pM) for Py-6. Separation of immunoglobulin G (IgG), human serum albumin, lipase, and myoglobin after labeling with Py-6 were performed. The method was further modified to make it amenable to automation. Unlike many other amino reactive reagents used to label protein amino groups, reaction with Py-1 and Py-6 do not alter the charge of the protein and the advantage of this with respect to electrophoretic separations is discussed.