The term kinetic stability is used to describe proteins that are trapped in a specific conformation because of an unusually high-unfolding barrier that results in very slow unfolding rates. Motivated by the observation that some proteins are resistant to sodium dodecyl sulfate (SDS)-induced denaturation, an attempt was made to determine whether this property is a result of kinetic stability. We studied many proteins, including a few kinetically stable proteins known to be resistant to SDS. The resistance to SDS-induced denaturation was investigated by comparing the migration on polyacrylamide gels of identical boiled and unboiled protein samples containing SDS. On the basis of the different migration of these samples, eight proteins emerged as being resistant to SDS. The kinetic stability of these proteins was confirmed by their slow unfolding rate upon incubation in guanidine hydrochloride. Further studies showed that these proteins were also extremely resistant to proteolysis by proteinase K, suggesting that a common mechanism may account for their resistance to SDS and proteolytic cleavage. Together, these observations suggest that a rigid protein structure may be the physical basis for kinetic stability and that resistance to SDS may serve as a simple assay for identifying proteins whose native conformations are kinetically trapped. Remarkably, most of the kinetically stable SDS-resistant proteins in this study are oligomeric beta-sheet proteins, suggesting a bias of these types of structures toward kinetic stability.

SummaryThe TonB system of Escherichia coli (TonB/ExbB/ ExbD) transduces the protonmotive force (pmf) of the cytoplasmic membrane to drive active transport by high-affinity outer membrane transporters. In this study, chromosomally encoded ExbD formed formaldehyde-linked complexes with TonB, ExbB and itself (homodimers) in vivo. Pmf was required for detectable cross-linking between TonB-ExbD periplasmic domains. Consistent with that observation, the presence of inactivating transmembrane domain mutations ExbD(D25N) or TonB(H20A) also prevented efficient formaldehyde cross-linking between ExbD and TonB. A specific site of periplasmic interaction occurred between ExbD(A92C) and TonB(A150C) and required functional transmembrane domains in both proteins. Conversely, neither TonB, ExbB nor pmf were required for ExbD dimer formation. These data suggest two possible models where either dynamic complex formation occurred through transmembrane domains or the transmembrane domains of ExbD and TonB configure their respective periplasmic domains. Analysis of T7-tagged ExbD with anti-ExbD antibodies revealed that a T7 tag was responsible both for our previous failure to detect T7-ExbD-ExbB and T7-ExbD-TonB formaldehyde-linked complexes and for the concomitant artefactual appearance of T7-ExbD trimers.

Most proteins are in equilibrium with partially and globally unfolded conformations. In contrast, kinetically stable proteins (KSPs) are trapped by an energy barrier in a specific state, unable to transiently sample other conformations. Among many potential roles, it appears that kinetic stability (KS) is a feature used by nature to allow proteins to maintain activity under harsh conditions and to preserve the structure of proteins that are prone to misfolding. The biological and pathological significance of KS remains poorly understood because of the lack of simple experimental methods to identify this property and its infrequent occurrence in proteins. Based on our previous correlation between KS and a protein's resistance to the denaturing detergent SDS, we show here the application of a diagonal 2D (D2D) SDS/PAGE assay to identify KSPs in complex mixtures. We applied this method to the lysate of Escherichia coli and upon proteomics analysis have identified 50 nonredundant proteins that were SDS-resistant (i.e., kinetically stable). Structural and functional analyses of a subset (44) of these proteins with known 3D structure revealed some potential structural and functional biases toward and against KS. This simple D2D SDS/PAGE assay will allow the widespread investigation of KS, including the proteomics-level identification of KSPs in different systems, potentially leading to a better understanding of the biological and pathological significance of this intriguing property of proteins.electrophoresis ͉ kinetic trap ͉ proteomics ͉ sodium dodecyl sulfate ͉ stability K inetic stability (KS) refers to the presence of an unusually high energy barrier that traps proteins in their native conformations, thereby dramatically decreasing their unfolding rate under a variety of conditions (1). This barrier allows kinetically stable proteins (KSPs) to maintain their native fold and activity for longer periods of time than would be expected for typical proteins, even in harsh environments. This unique feature is likely to play important biological roles, including the regulation of protein turnover, the protection of some proteins from proteolysis (1), and the entrapment of aggregation-prone proteins in their native state (2). Perhaps the best examples to illustrate the importance of KS are the proteins ␣-lytic protease and transthyretin (TTR). ␣-Lytic protease is synthesized with a pro-region that is essential for folding (1). Upon folding, ␣-lytic protease becomes active and cleaves off the pro region, leaving itself kinetically trapped in an active metastable conformation that is less stable than the unfolded state (3). In the case of TTR, the high thermodynamic and kinetic stability (KS) of the native tetramer is often compromised by mutation, leading to the population of a monomeric conformation that is the precursor of the aggregated species linked to the disease familial amyloid polyneuropathy (4). The large difference in the thermodynamic stability of ␣-lytic protease and TTR shows that KS need not correlate to th...

The TonB system of Gram-negative bacteria provides passage across the outer membrane (OM) diffusion barrier that otherwise limits access to large, scarce, or important nutrients. In Escherichia coli, the integral cytoplasmic membrane (CM) proteins TonB, ExbB, and ExbD couple the CM proton motive force (PMF) to active transport of iron-siderophore complexes and vitamin B 12 across the OM through high-affinity transporters. ExbB is an integral CM protein with three transmembrane domains. The majority of ExbB occupies the cytoplasm. Here, the importance of the cytoplasmic ExbB carboxy terminus (residues 195 to 244) was evaluated by cysteine scanning mutagenesis. D211C and some of the substitutions nearest the carboxy terminus spontaneously formed disulfide cross-links, even though the cytoplasm is a reducing environment. ExbB N196C and D211C substitutions were converted to Ala substitutions to stabilize them. Only N196A, D211A, A228C, and G244C substitutions significantly decreased ExbB activity. With the exception of ExbB(G244C), all of the substituted forms were dominant. Like wild-type ExbB, they all formed a formaldehyde cross-linked tetramer, as well as a tetramer cross-linked to an unidentified protein(s). In addition, they could be formaldehyde cross-linked to ExbD and TonB. Taken together, the data suggested that they assembled normally. Three of four ExbB mutants were defective in supporting both the PMF-dependent formaldehyde cross-link between the periplasmic domains of TonB and ExbD and the proteinase K-resistant conformation of TonB. Thus, mutations in a cytoplasmic region of ExbB prevented a periplasmic event and constituted evidence for signal transduction from cytoplasm to periplasm in the TonB system.

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