Characterizing the Range of Extracellular Protein Post-Translational Modifications in a Cellulose-Degrading Bacteria Using a Multiple Proteolyic Digestion/Peptide Fragmentation Approach

Abstract

Post-translational modifications (PTMs) are known to play a significant role in many biological functions. The focus of this study is to optimize an integrated experimental/informatics approach to more confidently characterize the range of post-translational modifications of the cellulosome protein complex used by the bacterium Clostridium thermocellum to better understand how this protein machine is tuned for enzymatic cellulose solubilization. To enhance comprehensive characterization, the extracellular cellulosome proteins were analyzed using multiple proteolytic digests (trypsin, Lys-C, Glu-C) and multiple fragmentation techniques (collisionally activated dissociation, electron transfer dissociation, decision tree). As expected, peptide and protein identifications were increased by utilizing alternate proteases and fragmentation methods, in addition to the increase in protein sequence coverage. The complementarity of these experiments also allowed for a global exploration of PTMs associated with the cellulosome based upon a set of defined PTMs that included methylation, oxidation, acetylation, phosphorylation, and signal peptide cleavage. In these experiments, 85 modified peptides corresponding to 28 cellulosome proteins were identified. Many of these modifications were located in active cellulolytic or structural domains of the cellulosome proteins, suggesting a level of possible regulatory control of protein function in various cellulotyic conditions. The use of complementary proteolytic digestion/peptide fragmentation processes allowed for independent verification of PTMs in different experiments, thus leading to increased confidence in PTM identifications.

Determination of the native features of the exoglucanase Cel48S from Clostridium thermocellum

“…The structure of the ctCel48S_CD proteins showed no posttranslational modifications. A previous mass spectrum analysis of the cellulosome components revealed some potential modifications of rCel48S, including the oxidation of the M290, M451, M552, W266, W493, W469, M298, W472, W595, and Y265 residues [ 54 ]. These modifications were likely caused by oxidative damage during sample preparation.…”

Arming the troops: Post-translational modification of extracellular bacterial proteins

“…This PTM is postulated to promote protein flexibility, whereas methylation of glutamic acid may aid cellulosome localisation. 10 Studies closely scrutinising secreted protein methylation are limited. However, outer membrane proteins have been studied in greater detail.…”

“…5 Consequently, independent approaches are often required to validate these ''high throughput'' technologies. 2,10 These include structural and functional studies, although as always, these too can be challenging. 11 Although it is now widely accepted that PTMs occur in bacteria, most studies have focused on cell-associated protein modifications, and relatively few have considered the modifications associated with proteins secreted into the extracellular milieu.…”

The Russian Doll Model: How Bacteria Shape Successful and Sustainable Inter-Kingdom Relationships

“…The concept of protein species (Jungblut et al, 2008), firstly introduced to avoid confusion with the term "isozyme" (that mean the product of a different gene encoding a protein with analogous function, e.g., catalyzing the same reaction with different efficacy and sometimes different direction), refers to different PT modified proteins and is now well accepted. There is an increasing evidence that PTMs have a huge biological significance because they determine peculiar protein properties like half-life, interaction with other proteins, cellular localization (Kobir et al, 2011) as well as diversified protein functions such as redox signaling, enzyme activity, chemotaxis, and metabolic flux tuning (Dykstra et al, 2013). Actually, each protein species (for instance a protein bearing glycosylation) can solve a specific physiological function not performed by the same gene product that has been processed differently (for instance the same protein when phosphorylated).…”

“…Actually, each protein species (for instance a protein bearing glycosylation) can solve a specific physiological function not performed by the same gene product that has been processed differently (for instance the same protein when phosphorylated). As an example, methylations or acetylations of cellulosome scaffold proteins have critical functional significance, by impacting differently cellulose utilization efficiency (Dykstra et al, 2013). In this way, only one gene can support several tasks thus guarantee economy of genes and of energy required for transcription and translation (Figure 1).…”