Protein Life Cycle Research Articles

Proteomic and Metabolic Analysis of Pinus halepensis Mill. Embryonal Masses Induced under Heat Stress.

Understanding the physiological and molecular adjustments occurring during tree stress response is of great importance for forest management and breeding programs. Somatic embryogenesis has been used as a model system to analyze various processes occurring during embryo development, including stress response mechanisms. In addition, "priming" plants with heat stress during somatic embryogenesis seems to favor the acquisition of plant resilience to extreme temperature conditions. In this sense, Pinus halepensis somatic embryogenesis was induced under different heat stress treatments (40 °C for 4 h, 50 °C for 30 min, and 60 °C for 5 min) and its effects on the proteome and the relative concentration of soluble sugars, sugar alcohols and amino acids of the embryonal masses obtained were assessed. Heat severely affected the production of proteins, and 27 proteins related to heat stress response were identified; the majority of the proteins with increased amounts in embryonal masses induced at higher temperatures consisted of enzymes involved in the regulation of metabolism (glycolysis, the tricarboxylic acid cycle, amino acid biosynthesis and flavonoids formation), DNA binding, cell division, transcription regulation and the life-cycle of proteins. Finally, significant differences in the concentrations of sucrose and amino acids, such as glutamine, glycine and cysteine, were found.

Diblock copolymers enhance folding of a mechanosensitive membrane protein during cell-free expression

The expression and integration of membrane proteins into vesicle membranes is a critical step in the design of cell-mimetic biosensors, bioreactors, and artificial cells. While membrane proteins have been integrated into a variety of nonnatural membranes, the effects of the chemical and physical properties of these vesicle membranes on protein behavior remain largely unknown. Nonnatural amphiphiles, such as diblock copolymers, provide an interface that can be synthetically controlled to better investigate this relationship. Here, we focus on the initial step in a membrane protein's life cycle: expression and folding. We observe improvements in both the folding and overall production of a model mechanosensitive channel protein, the mechanosensitive channel of large conductance, during cell-free reactions when vesicles containing diblock copolymers are present. By systematically tuning the membrane composition of vesicles through incorporation of a poly(ethylene oxide)-b-poly(butadiene) diblock copolymer, we show that membrane protein folding and production can be improved over that observed in traditional lipid vesicles. We then reproduce this effect with an alternate membrane-elasticizing molecule, C12E8 Our results suggest that global membrane physical properties, specifically available membrane surface area and the membrane area expansion modulus, significantly influence the folding and yield of a membrane protein. Furthermore, our results set the stage for explorations into how nonnatural membrane amphiphiles can be used to both study and enhance the production of biological membrane proteins.

Single-residue posttranslational modification sites at the N-terminus, C-terminus or in-between: To be or not to be exposed for enzyme access.

Many protein posttranslational modifications (PTMs) are the result of an enzymatic reaction. The modifying enzyme has to recognize the substrate protein's sequence motif containing the residue(s) to be modified; thus, the enzyme's catalytic cleft engulfs these residue(s) and the respective sequence environment. This residue accessibility condition principally limits the range where enzymatic PTMs can occur in the protein sequence. Non‐globular, flexible, intrinsically disordered segments or large loops/accessible long side chains should be preferred whereas residues buried in the core of structures should be void of what we call canonical, enzyme‐generated PTMs. We investigate whether PTM sites annotated in UniProtKB (with MOD_RES/LIPID keys) are situated within sequence ranges that can be mapped to known 3D structures. We find that N‐ or C‐termini harbor essentially exclusively canonical PTMs. We also find that the overwhelming majority of all other PTMs are also canonical though, later in the protein's life cycle, the PTM sites can become buried due to complex formation. Among the remaining cases, some can be explained (i) with autocatalysis, (ii) with modification before folding or after temporary unfolding, or (iii) as products of interaction with small, diffusible reactants. Others require further research how these PTMs are mechanistically generated in vivo.