Protein quinary interactions organize the cellular interior and its metabolism. Although the interactions stabilizing secondary, tertiary, and quaternary protein structure are well defined, details about the protein-matrix contacts that comprise quinary structure remain elusive. This gap exists because proteins function in the crowded cellular environment, but are traditionally studied in simple buffered solutions. We use NMR-detected H/D exchange to quantify quinary interactions between the B1 domain of protein G and the cytosol of Escherichia coli. We demonstrate that a surface mutation in this protein is 10-fold more destabilizing in cells than in buffer, a surprising result that firmly establishes the significance of quinary interactions. Remarkably, the energy involved in these interactions can be as large as the energies that stabilize specific protein complexes. These results will drive the critical task of implementing quinary structure into models for understanding the proteome.T he inside of cells is packed with macromolecules whose concentrations reach 300-400 g/L (1). Compared with the ideal (dilute) environments conventionally used to study proteins, crowding inside cells can significantly alter the biophysical landscape of proteins, including their equilibrium thermodynamic stability (2-6). Experimental and computational efforts establish that crowding effects arise from a combination of short-range (steric) repulsions and longer-range (often called soft) interactions between macromolecules (7-13). Despite a growing number of incell protein studies (2-6), there is no quantitative information about the energetics of quinary interactions.Amide proton exchange experiments have been used for more than 50 y to measure equilibrium protein stability, defined as the Gibbs free energy required to open the protein and expose individual backbone amide protons to solvent, ΔG°′ op (14). For the B1 domain of protein G (GB1), ΔG°′ op equals −RTln(k obs /k uns ), where R is the gas constant, T is the absolute temperature, k obs is the observed rate of exchange, and k uns is the rate in an unstructured peptide (6). We know that the cytoplasm does not affect k uns (15). Most importantly, we know that for exchange under these conditions ΔG°′ op approximates the free energy required to denature the protein, ΔG°′ den (6). Therefore, these experiments provide a thermodynamically rigorous measure of equilibrium global protein stability. Using this information, we quantified the stability of GB1 at the residue level in Escherichia coli (6) via NMRdetected backbone amide hydrogen/deuterium exchange (16).Thermodynamic cycles (17) can be used to quantify the energetics of interactions between proteins in specific protein complexes (17,18) and between side chains in globular proteins (19,20). Briefly, the individual effects of two single-site amino acid changes are compared with the combined effect of both mutations. If the sites interact, the sum of the contributions from the single-site changes will not equal the contributi...

Fluorine-containing amino acids are valuable probes for the biophysical characterization of proteins. Current methods for (19)F-labeled protein production involve time-consuming genetic manipulation, compromised expression systems and expensive reagents. We show that Escherichia coli BL21, the workhorse of protein production, can utilise fluoroindole for the biosynthesis of proteins containing (19)F-tryptophan.

Significance Proteins function in a sea of macromolecules within cells, but are traditionally studied under ideal conditions in vitro . The more details we amass from experiments performed in cells, the closer we will get to understanding fundamental aspects of protein chemistry in the cellular environment. In addition to furthering our essential knowledge of biochemistry, advancements in the field of macromolecular crowding will drive efforts to stabilize protein-based therapeutics. Here, we show that protein stability can be measured at the residue level in living cells without adding destabilizing cosolutes or heat.

No abstract

Electrospray ionization (ESI) mass spectrometry (MS) has proven to be an extremely powerful technique for studying the stoichiometry and binding strength of peptide-metal complexes. We have found a significant new problem in the ESI-MS of zinc-peptide systems involving the deposition of zinc in the ESI emitter. This deposition of zinc during the experiment removes a significant amount of zinc ions from the solution, impacting the resulting mass spectral intensities used to quantify the amount of the zinc-bound species. Analysis of infused zinc-peptide samples with atomic absorption spectrometry and with a custom-built nanoflow ESI source confirms the alteration of the analyte solutions with positive or negative or no potential applied to the emitter. Ultimately, the location of the zinc deposition was determined to be the stainless steel emitter. The use of a custom-built nanoESI interface using glass emitters was found to mitigate the zinc deposition problem. The phenomenon of metal deposition warrants further investigation as it may not be limited to just zinc and may represent a significant obstacle in the ESI-MS analysis of all protein-metal systems.

Prothymosin-alpha is a highly acidic protein consisting of 110 amino acids. The central segment of this protein, residues 51-89, is thought to be involved in metal binding which may be necessary for its physiological function. To carry out studies of this peptide, this central segment was synthesized in a linear fashion using Fmoc-based methods on rink amide MBHA resin. However, this peptide could not be purified with the typical straightforward approach of RP HPLC followed by negative mode electrospray ionization mass spectrometry (ESI-MS). This was attributed to the high proportion of acidic residues: 26 out of the 39 residues are aspartic and glutamic acids. The acidity of the peptide prevented retention on the RP HPLC column. Additionally, the ability of the highly negatively charged peptide to retain sodium ions prevented molecular weight determination with ESI-MS. A systematic approach to the purification of this highly acidic peptide was undertaken. Ultimately, strong anion exchange chromatography was used to purify the peptide. Extensive desalting using dialysis was required prior to ESI-MS, and the choice of the buffer proved to be critical. In the end, a purification method was devised that yielded a highly purified peptide and is readily compatible with analysis by ESI-MS.

Prothymosin-α is a small, multifunctional intrinsically disordered protein associated with cell survival and proliferation which binds multiple Zn ions and undergoes partial folding. The interaction between prothymosin-α and at least two of its protein targets is significantly enhanced in the presence of Zn ions, suggesting that Zn binding plays a role in the protein's function. The primary sequence of prothymosin-α is highly acidic, with almost 50% comprised of Asp and Glu, and is unusual for a Zn-binding protein as it lacks Cys and His residues. To gain a better understanding of the nature of the Zn-prothymosin-α interactions and the protein's ability to discriminate Zn over other divalent cations (e.g., Ca, Co, Mg) we synthesized a set of three model peptides and characterized the effect of metal binding using electrospray ionization mass spectrometry (ESI MS) and circular dichroism (CD) spectroscopy. ESI MS data reveal that the native peptide model of the glutamic acid rich region binds 4 Zn ions with apparent, stepwise K values that are, at highest, in the tens of micromolar range. A peptide model with the same amino acid composition as the native sequence, but with the residues arranged randomly, showed no evidence of structural change by CD upon introduction of Zn. These results suggest that the high net negative charge of the glutamic acid-rich region of prothymosin-α is not a sufficient criterion for Zn to induce a structural change; rather, Zn binding to prothymosin-α is sequence specific, providing important insight into the behavior of intrinsically disordered proteins.

Temperature experiments show a two-step unfolding of the protein. Above 45 C we have molten globule kind of loosened structure, and the complete unfolding occurs above 70 C. The unfolding is accompanied with aggregation of the protein. The aggregation can however be prevented by relatively low pressure of 0.2 GPa. Using the above phase transition points the T-p phase diagram of parvalbumin was determined in presence of Ca 2þ . It shows a very complex pattern, containing two different molten globule phases besides the native, unfolded and aggregated ones.