(15) After quenching PK, proteins are then fully trypsinized and prepared for analysis by liquid chromatography/tandem mass spectrometry (LC-MS/MS). A low level of protease, active for a brief period of time (1 min), ensures that PK cleaves only at exposed or unstructured sites of target proteins, thereby encoding structural information on the protein’s conformation into cleavage sites. Following a period of time, the structures of the proteins in these complex mixtures are probed by subjecting them to pulse proteolysis with proteinase K (PK). (14) Clarified extracts are then divided so that one portion is retained in its native state, and a separate portion is first unfolded by addition of high concentrations of chemical denaturants (6 M guanidinium chloride (GdmCl)) and then refolded by lowering the denaturant concentration (by dilution or by dialysis). We chose to lyse cells in this way because it retains the vast majority of proteins in their native state, including weakly bound structures (13) and even polysome-nascent chain complexes. coli cells (type strain K-12) are grown in a defined media to the end of log phase (OD 0.8), resuspended in a lysis buffer, frozen by immersion into liquid nitrogen, and lysed by cryogenic pulverization (see Materials and Methods). In our experimental design ( Figure 1), E. These experiments have greater structural resolution than classic spectroscopic measures (such as circular dichroism, fluorescence, or FRET) employed in many protein folding studies and reveal that following denaturation, many proteins are incapable of fully returning to their native structures on physiological time scales under a standard set of in vitro refolding conditions. Using mass spectrometry, we analyze the digestion patterns to globally assess structural differences between native proteins and their “refolded” forms. coli lysates under conditions without precipitation and then interrogating the resulting protein structures using a permissive protease that preferentially cleaves at flexible regions. We accomplish this by first unfolding and refolding E. In this study, we introduce an experimental approach to probe protein refolding kinetics for whole proteomes. In any case, whether nonrefoldability is common for more complex “nonmodel” proteins is not generally known. (11,12) In other situations, it may be because the native state is challenging to access or is metastable relative to other folded (but non-native) conformations. In the unusual case of alpha-lytic protease, it is because the unfolded state is more stable than the native state. (6−10) There are several possible reasons why a protein might not be able to refold to its native form. (4,5) These studies are typically interpreted through the ground truth of Anfinsen’s dogma, which states that proteins can intrinsically refold to their native states from unfolded forms because the native states represent global thermodynamic minima. Many experiments of protein folding, conducted on purified, small, single-domain, soluble proteins, follow the proportion of protein molecules that are folded as a function of time, temperature, denaturant concentration, or sequence (1−3) and have yielded immense insight into the molecular determinants that underpin stable globular folds.
Hence, these results illuminate when exogenous factors and processes, such as chaperones or cotranslational folding, might be required for efficient protein folding. We also identify several properties and fold-types that are correlated with slow refolding on the minute time scale. coli proteome is not intrinsically refoldable on physiological time scales, a cohort that is enriched with certain fold-types, domain organizations, and other biophysical features. coli proteome expressed during log-phase growth, and among this group, we find that one-third of the E. Our study covers the majority of the soluble E. Here, we introduce an experimental approach to probe protein refolding kinetics for whole proteomes using mass spectrometry-based proteomics. However, these studies have generally not been representative of the complexity of natural proteomes, which consist of many proteins with complex architectures and domain organizations. Decades of research on protein folding have primarily focused on a subset of small proteins that can reversibly refold from a denatured state.
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