Protein interactions in physiological environments

The interactions of three proteins (ΔTat-GB1, cytochrome c and flavodoxin) were explored in Escherichia coli cells and extracts. The results indicate features of the physicochemical basis for protein assembly in the complex cytoplasm. Some of this work featured as a highlighted article in Protein Science. Although key principles of molecular recognition have been gleaned from ‘reductionist’ studies performed in dilute solution, physiologically-relevant insights require protein characterisation under native or near-native conditions. In particular, knowledge of the low specificity ‘quinary’ interactions that dynamically organise the cytoplasm is scarce. In-cell NMR indicated that all three proteins interact pervasively with the cytoplasm, despite their different sizes and surface properties. NMR, size exclusion chromatography (SEC) and/or native gel electrophoresis was used to probe the physicochemical basis for the interactions of each test protein in E. coli extracts. The charge of cytochrome c and four of its mutants was determined by membrane confined electrophoresis. These findings indicate the effects of decreased charge on cytochrome c interactions in extracts. The weak, low-specificity cytochrome c-flavodoxin complex was studied in volume-occupied solutions to dissect the effectsof the cytoplasmic architectures on this ‘quinary-like’ interaction. The intricate interplay of charge, hydrophobic interactions and preferential hydration in governing cytoplasmic interactions is emphasised. These findings provide fresh evidence in support of a physicochemical model for cytoplasmic structuring. A novel strategy for 19F tryptophan incorporation in E. coli was also developed which involved the addition of 30-60 mg/L of the tryptophan precursor 5-fluoroindole to the growth medium. 19F tryptophan-labelled flavodoxin and GB1 were subsequently studied by 19F NMR in E. coli or extracts.2017-06-0

Protein interactions in physiological environments

The interactions of three proteins (ΔTat-GB1, cytochrome c and flavodoxin) were explored in Escherichia coli cells and extracts. The results indicate features of the physicochemical basis for protein assembly in the complex cytoplasm. Some of this work featured as a highlighted article in Protein Science. Although key principles of molecular recognition have been gleaned from ‘reductionist’ studies performed in dilute solution, physiologically-relevant insights require protein characterisation under native or near-native conditions. In particular, knowledge of the low specificity ‘quinary’ interactions that dynamically organise the cytoplasm is scarce. In-cell NMR indicated that all three proteins interact pervasively with the cytoplasm, despite their different sizes and surface properties. NMR, size exclusion chromatography (SEC) and/or native gel electrophoresis was used to probe the physicochemical basis for the interactions of each test protein in E. coli extracts. The charge of cytochrome c and four of its mutants was determined by membrane confined electrophoresis. These findings indicate the effects of decreased charge on cytochrome c interactions in extracts. The weak, low-specificity cytochrome c-flavodoxin complex was studied in volume-occupied solutions to dissect the effectsof the cytoplasmic architectures on this ‘quinary-like’ interaction. The intricate interplay of charge, hydrophobic interactions and preferential hydration in governing cytoplasmic interactions is emphasised. These findings provide fresh evidence in support of a physicochemical model for cytoplasmic structuring. A novel strategy for 19F tryptophan incorporation in E. coli was also developed which involved the addition of 30-60 mg/L of the tryptophan precursor 5-fluoroindole to the growth medium. 19F tryptophan-labelled flavodoxin and GB1 were subsequently studied by 19F NMR in E. coli or extracts.2017-06-0

Grasping the nature of the cell interior: fromphysiological chemistrytochemical biology

Current models of the cell interior emphasise its crowded, chemically complex and dynamically organised structure. Although the chemical composition of cells is known, the cooperative intermolecular interactions that govern cell ultrastructure are poorly understood. A major goal of biochemistry is to capture these myriad interactions in vivo. We consider the landmark discoveries that have shaped this objective, starting from the vitalist framework established by early natural philosophers. Through this historical revisionism, we extract important lessons for the bioinspired chemists of today. Scientific specialisation tends to insulate seminal ideas and hamper the unification of paradigms across biology. Therefore, we call for interdisciplinary collaboration in grappling with the complex cell interior. Recent successes in integrative structural biology and chemical biology demonstrate the power of hybrid approaches. The future roles of the (bio)chemist and model systems are also discussed as starting points for in vivo explorations

Grasping the nature of the cell interior: fromphysiological chemistrytochemical biology

Current models of the cell interior emphasise its crowded, chemically complex and dynamically organised structure. Although the chemical composition of cells is known, the cooperative intermolecular interactions that govern cell ultrastructure are poorly understood. A major goal of biochemistry is to capture these myriad interactions in vivo. We consider the landmark discoveries that have shaped this objective, starting from the vitalist framework established by early natural philosophers. Through this historical revisionism, we extract important lessons for the bioinspired chemists of today. Scientific specialisation tends to insulate seminal ideas and hamper the unification of paradigms across biology. Therefore, we call for interdisciplinary collaboration in grappling with the complex cell interior. Recent successes in integrative structural biology and chemical biology demonstrate the power of hybrid approaches. The future roles of the (bio)chemist and model systems are also discussed as starting points for in vivo explorations

Short Arginine Motifs Drive Protein Stickiness in the <i>Escherichia coli</i> Cytoplasm

Although essential to numerous biotech applications, knowledge of molecular recognition by arginine-rich motifs in live cells remains limited. ¹H,¹⁵N HSQC and ¹⁹F NMR spectroscopies were used to investigate the effects of C-terminal −GR_<i>n</i> (<i>n</i> = 1–5) motifs on GB1 interactions in Escherichia coli cells and cell extracts. While the “biologically inert” GB1 yields high-quality in-cell spectra, the −GR_<i>n</i> fusions with <i>n</i> = 4 or 5 were undetectable. This result suggests that a tetra-arginine motif is sufficient to drive interactions between a test protein and macromolecules in the E. coli cytoplasm. The inclusion of a 12 residue flexible linker between GB1 and the −GR₅ motif did not improve detection of the “inert” domain. In contrast, all of the constructs were detectable in cell lysates and extracts, suggesting that the arginine-mediated complexes were weak. Together these data reveal the significance of weak interactions between short arginine-rich motifs and the E. coli cytoplasm and demonstrate the potential of such motifs to modify protein interactions in living cells. These interactions must be considered in the design of (<i>in vivo</i>) nanoscale assemblies that rely on arginine-rich sequences

Specific ion effects on macromolecular interactions inescherichia coliextracts

Protein characterization in situ remains a major challenge for protein science. Here, the interactions of Tat-GB1 in Escherichia coli cell extracts were investigated by NMR spectroscopy and size exclusion chromatography (SEC). Tat-GB1 was found to participate in high molecular weight complexes that remain intact at physiologically-relevant ionic strength. This observation helps to explain why Tat-GB1 was not detected by in-cell NMR spectroscopy. Extracts pre-treated with RNase A had a different SEC elution profile indicating that Tat-GB1 predominantly interacted with RNA. The roles of biological and laboratory ions in mediating macromolecular interactions were studied. Interestingly, the interactions of Tat-GB1 could be disrupted by biologically-relevant multivalent ions. The most effective shielding of interactions occurred in Mg2+-containing buffers. Moreover, a combination of RNA digestion and Mg2+ greatly enhanced the NMR detection of Tat-GB1 in cell extracts

Protein charge determination and implications for interactions in cell extracts

Decades of dilute-solution studies have revealed the influence of charged residues on protein stability, solubility and stickiness. Similar characterizations are now required in physiological solutions to understand the effect of charge on protein behavior under native conditions. Toward this end, we used free boundary and native gel electrophoresis to explore the charge of cytochrome c in buffer and in Escherichia coli extracts. We find that the charge of cytochrome c was approximate to 2-fold lower than predicted from primary structure analysis. Cytochrome c charge was tuned by sulfate binding and was rendered anionic in E. coli extracts due to interactions with macroanions. Mutants in which three or four cationic residues were replaced with glutamate were charge-neutral and inert in extracts. A comparison of the interaction propensities of cytochrome c and the mutants emphasizes the role of negative charge in stabilizing physiological environments. Charge-charge repulsion and preferential hydration appear to prevent aggregation. The implications for molecular organization in vivo are discussed

Protein charge determination and implications for interactions in cell extracts

Decades of dilute-solution studies have revealed the influence of charged residues on protein stability, solubility and stickiness. Similar characterizations are now required in physiological solutions to understand the effect of charge on protein behavior under native conditions. Toward this end, we used free boundary and native gel electrophoresis to explore the charge of cytochrome c in buffer and in Escherichia coli extracts. We find that the charge of cytochrome c was approximate to 2-fold lower than predicted from primary structure analysis. Cytochrome c charge was tuned by sulfate binding and was rendered anionic in E. coli extracts due to interactions with macroanions. Mutants in which three or four cationic residues were replaced with glutamate were charge-neutral and inert in extracts. A comparison of the interaction propensities of cytochrome c and the mutants emphasizes the role of negative charge in stabilizing physiological environments. Charge-charge repulsion and preferential hydration appear to prevent aggregation. The implications for molecular organization in vivo are discussed

Physicochemical Properties of Cells and Their Effects on Intrinsically Disordered Proteins (IDPs)

It has long been axiomatic that a protein’s structure determines its function. Intrinsically disordered proteins (IDPs) and disordered protein regions (IDRs) defy this structure–function paradigm. They do not exhibit stable secondary and/or tertiary structures and exist as dynamic ensembles of interconverting conformers with preferred, nonrandom orientations.(1-4) The concept of IDPs and IDRs as functional biological units was initially met with skepticism. For a long time, disorder, intuitively implying chaos, had no place in our perception of orchestrated molecular events controlling cell biology. Over the past years, however, this notion has changed. Aided by findings that structural disorder constitutes an ubiquitous and abundant biological phenomenon in organisms of all phyla,(5-7) and that it is often synonymous with function,(8-11) disorder has become an integral part of modern protein biochemistry. Disorder thrives in eukaryotic signaling pathways(12) and functions as a prominent player in many regulatory processes.(13-15) Disordered proteins and protein regions determine the underlying causes of many neurodegenerative disorders and constitute the main components of amyloid fibrils.(16) They further contribute to many forms of cancer, diabetes and to cardiovascular and metabolic diseases.(17, 18) Research into disordered proteins produced significant findings and established important new concepts. On the structural side, novel experimental and computational approaches identified and described disordered protein ensembles(3, 19, 20) and led to terms such as secondary structure propensities, residual structural features, and transient long-range contacts.(1, 21) The discovery of coupled folding-and-binding reactions defined the paradigm of disorder-to-order transitions(22) and high-resolution insights into the architectures of amyloid fibrils were obtained.(23, 24) On the biological side, we learned about the unexpected intracellular stability of disordered proteins, their roles in integrating post-translational protein modifications in cell signaling and about their functions in regulatory processes ranging from transcription to cell fate decisions.(15, 25, 26) One open question remaining to be addressed is how these in vitro structural insights relate to biological in vivo effects. How do complex intracellular environments modulate the in vivo properties of disordered proteins and what are the implications for their biological functions (Figure 1)?(27-29