R. John Ellis

Macromolecular crowding: obvious but underappreciated

Biological macromolecules evolve and function within intracellular environments that are crowded with other macromolecules. Crowding results in surprisingly large quantitative effects on both the rates and the equilibria of interactions involving macromolecules, but such interactions are commonly studied outside the cell in uncrowded buffers. The addition of high concentrations of natural and synthetic macromolecules to such buffers enables crowding to be mimicked in vitro, and should be encouraged as a routine variable to study. The stimulation of protein aggregation by crowding might account for the existence of molecular chaperones that combat this effect. Positive results of crowding include enhancing the collapse of polypeptide chains into functional proteins, the assembly of oligomeric structures and the efficiency of action of some molecular chaperones and metabolic pathways.

The most abundant protein in the world

Abstract The most abundant protein in nature is probably the chloroplast enzyme ribulose bisphosphate carboxylase/oxygenase (Fraction I protein). It is arguably the most important enzyme because it catalyses the carbon dioxide-fixing step in photosynthesis. The synthesis of this protein depends upon the interaction of nucleargenes with the genetic system located in the chloroplast, and involves the transfer of polypeptides across the chloroplast envelope by a post-translational mechanism.

The Transport of Proteins into Chloroplasts

We have partially purified a soluble protease from Pisum sativum chloroplasts involved in the processing of precursor polypeptides imported into the organelle. The enzyme processes precursors of both stromal and thylakoid proteins to the mature size, but is inactive against all proteins so far tested other than precursors destined for the chloroplast. The enzyme processes precursors from wheat and barley, and is therefore not species-specific. It has a relative molecular mass of about 180 000 and a pH optimum near 9. The enzyme is inhibited by ethylenediamine tetraacetate and 1,10-phenanthroline but not by serine- or thiol-protease inhibitors.

Molecular chaperones: proteins essential for the biogenesis of some macromolecular structures

Many polypeptides can self-assemble into functional structures while others assemble only in the presence of additional proteins (molecular chaperones) which are not components of the final structure. We discuss here the effect that the recognition of the essential roles played by these proteins in assembly processes may have on the principle of spontaneous self-assembly.

Effects of macromolecular crowding on protein folding and aggregation.

We have studied the effects of polysaccharide and protein crowding agents on the refolding of oxidized and reduced hen lysozyme in order to test the prediction that association constants of interacting macromolecules in living cells are greatly increased by macromolecular crowding relative to their values in dilute solutions. We demonstrate that whereas refolding of oxidized lysozyme is hardly affected by crowding, correct refolding of the reduced protein is essentially abolished due to aggregation at high concentrations of crowding agents. The results show that the protein folding catalyst protein disulfide isomerase is particularly effective in preventing lysozyme aggregation under crowded conditions, suggesting that crowding enhances its chaperone activity. Our findings suggest that the effects of macromolecular crowding could have major implications for our understanding of how protein folding occurs inside cells.

Protein aggregation in crowded environments

Abstract The generic tendency of proteins to aggregate into non-functional, and sometimes cytotoxic, structures poses a universal problem for all types of cell. This tendency is greatly exacerbated by the high total concentration of macromolecules found within most intracellular compartments, a phenomenon referred to as macromolecular crowding. This review discusses the quantitative effects of crowding on protein aggregation and the role of molecular chaperones in combating this problem.

Protein synthesis in chloroplasts I. Light-driven synthesis of the large subunit of Fraction I protein by isolated pea chloroplasts

Abstract Intact isolated pea chloroplasts use light energy to incorporate labelled amino acids into protein. 25 % of this incorporation is present in a 150 000 × g chloroplast supernatant fraction. When this supernatant is analysed on sodium dodecyl sulphate polyacrylamide gels only one polypeptide is labelled. This polypeptide is the large subunit of Fraction I protein, a major protein constituent of the chloroplast. Identity of the soluble in vitro product with the large subunit of Fraction I protein was established by comparing a tryptic map of its [35S]methionine-labelled peptides with a tryptic map of the large subunit of Fraction I protein labelled in vivo with [35S]methionine. We conclude that only one of the many chloroplast soluble proteins, namely the large subunit of Fraction I protein, is synthesised on chloroplast ribosomes.

Protein synthesis in chloroplasts: II. Light-driven synthesis of membrane proteins by isolated pea chloroplasts

Abstract Washed membranes from pea chloroplasts can be resolved into at least 21 protein bands on sodium dodecylsulphate polyacrylamide gels, with molecular weights ranging from 13 000 to over 100 000. Isolated intact pea chloroplasts use both light energy and added ATP to incorporate [35S]methionine into six discrete membrane-bound products. Five of these are digestible by pronase, but not by ribonuclease, and have molecular weights of 85 000, 40 000, 32 000, 22 000 and 18 000. These proteins cannot be removed from the membranes by washing with water or EDTA, but can be solubilised by digitonin. The identity of these proteins is unknown, but they do not include cytochrome f , the coupling factor, or Photosystems I and II proteins.

Macromolecular crowding perturbs protein refolding kinetics: implications for folding inside the cell

We have studied the effects of macromolecular crowding on protein folding kinetics by studying the oxidative refolding of hen lysozyme in the absence and presence of high concentrations of bovine serum albumin and Ficoll 70. The heterogeneity characteristic of the lysozyme refolding process is preserved under crowded conditions. This, together with the observation that the refolding intermediates that accumulate to significant levels are very similar in the absence and presence of Ficoll, suggests that crowding does not alter substantially the energetics of the protein folding reaction. However, the presence of high concentrations of macromolecules results in the acceleration of the fast track of the refolding process whereas the slow track is substantially retarded. The results can be explained by preferential excluded volume stabilization of compact states relative to more unfolded states, and suggest that, relative to dilute solutions, the rates of many protein folding processes are likely to be altered under conditions that more closely resemble the intracellular environment.

Light-stimulated transcription of genes for two chloroplast polypeptides in isolated pea leaf nuclei.

Nuclei isolated from both light‐grown and dark‐grown leaves of Pisum sativum by Percoll density gradient centrifugation incorporate labelled UTP into RNA when supplemented with the other three nucleoside triphosphates. The RNA is heterodisperse, with transcripts up to at least 25S in size. Among these transcripts are sequences hybridizing to cloned DNA probes for wheat rRNA and two abundant chloroplast polypeptides of Pisum, viz. the small subunit of ribulose bisphosphate carboxylase and a polypeptide of the light‐harvesting chlorophyll a/b binding complex. Transcription of small subunit and light‐harvesting complex sequences is greater (18‐fold and 9‐fold, respectively) in nuclei from light‐grown leaves than in nuclei from dark‐grown leaves. Transcription of ribosomal genes, by contrast, is only doubled by growth in the light. Small subunit and light‐harvesting complex sequences transcribed in dark‐grown nuclei are not degraded in a 120 min chase. These results suggest that the stimulation of accumulation of small subunit and light‐harvesting complex mRNAs by exposure of Pisum seedlings to light is mediated by an increase in transcription rather than by a decrease in RNA degradation.