Unique Histidine Research Articles

Mg2+-dependent mechanism of environmental versatility in a multidrug efflux pump.

Tripartite resistance nodulation and cell division multidrug efflux pumps span the periplasm and are a major driver of multidrug resistance among Gram-negative bacteria. The periplasm provides a distinct environment between the inner and outer membranes of Gram-negative bacteria. Cations, such as Mg2+, become concentrated within the periplasm and, in contrast to the cytoplasm, its pH is sensitive to conditions outside the cell. Here, we reveal an interplay between Mg2+ and pH in modulating the dynamics of the periplasmic adaptor protein, AcrA, and its function within the prototypical AcrAB-TolC multidrug efflux pump from Escherichia coli. In the absence of Mg2+, AcrA becomes increasingly plastic within acidic conditions, but when Mg2+ is bound this is ameliorated, resulting in domain specific organisation in neutral to weakly acidic regimes. We establish a unique histidine residue directs these structural dynamics and is essential for sustaining pump efflux activity across acidic, neutral, and alkaline conditions. Overall, we propose Mg2+ conserves the structural mobility of AcrA to ensure optimal AcrAB-TolC function within rapid changing environments commonly faced by the periplasm during bacterial infection and colonization. This work highlights that Mg2+ is an important mechanistic component in this pump class and possibly across other periplasmic lipoproteins.

AlphaFold modeling of nepovirus 3C-like proteinases provides new insights into their diverse substrate specificities

The majority of picornaviral 3C proteinases (3Cpro) cleavage sites possess glutamine at the P1 position. Plant nepovirus 3C-like proteinases (3CLpro) show however much broader specificity, cleaving not only after glutamine, but also after several basic and hydrophobic residues. To investigate this difference, we employed AlphaFold to generate structural models of twelve selected 3CLpro, representing six substrate specificities. Generally, we observed favorable correlations between the architecture and charge of nepovirus proteinase S1 subsites and their ability to accept or restrict larger residues. The models identified a conserved aspartate residue close to the P1 residue in the S1 subsites of all nepovirus proteinases examined, consistent with the observed strong bias against negatively-charged residues at the P1 position of nepovirus cleavage sites. Finally, a cramped S4 subsite along with the presence of two unique histidine and serine residues explains the strict requirement of the grapevine fanleaf virus proteinase for serine at the P4 position.

ATP binding by an F1Fo ATP synthase ε subunit is pH dependent, suggesting a diversity of ε subunit functional regulation in bacteria.

It is a conjecture that the ε subunit regulates ATP hydrolytic function of the F1Fo ATP synthase in bacteria. This has been proposed by the ε subunit taking an extended conformation, with a terminal helix probing into the central architecture of the hexameric catalytic domain, preventing ATP hydrolysis. The ε subunit takes a contracted conformation when bound to ATP, thus would not interfere with catalysis. A recent crystallographic study has disputed this; the Caldalkalibacillus thermarum TA2.A1 F1Fo ATP synthase cannot natively hydrolyse ATP, yet studies have demonstrated that the loss of the ε subunit terminal helix results in an ATP synthase capable of ATP hydrolysis, supporting ε subunit function. Analysis of sequence and crystallographic data of the C. thermarum F1Fo ATP synthase revealed two unique histidine residues. Molecular dynamics simulations suggested that the protonation state of these residues may influence ATP binding site stability. Yet these residues lie outside the ATP/Mg2+ binding site of the ε subunit. We then probed the effect of pH on the ATP binding affinity of the ε subunit from the C. thermarum F1Fo ATP synthase at various physiologically relevant pH values. We show that binding affinity changes 5.9 fold between pH 7.0, where binding is weakest, to pH 8.5 where it is strongest. Since the C. thermarum cytoplasm is pH 8.0 when it grows optimally, this correlates to the ε subunit being down due to ATP/Mg2+ affinity, and not being involved in blocking ATP hydrolysis. Here, we have experimentally correlated that the pH of the bacterial cytoplasm is of critical importance for ε subunit ATP affinity regulated by second-shell residues thus the function of the ε subunit changes with growth conditions.

The unique histidine in OSCP subunit of F-ATP synthase mediates inhibition of the permeability transition pore by acidic pH.

The permeability transition pore (PTP) is a Ca2+-dependent mitochondrial channel whose opening causes a permeability increase in the inner membrane to ions and solutes. The most potent inhibitors are matrix protons, with channel block at pH 6.5. Inhibition is reversible, mediated by histidyl residue(s), and prevented by their carbethoxylation by diethylpyrocarbonate (DPC), but their assignment is unsolved. We show that PTP inhibition by H+ is mediated by the highly conserved histidyl residue (H112 in the human mature protein) of oligomycin sensitivity conferral protein (OSCP) subunit of mitochondrial F1FO (F)-ATP synthase, which we also show to undergo carbethoxylation after reaction of mitochondria with DPC. Mitochondrial PTP-dependent swelling cannot be inhibited by acidic pH in H112Q and H112Y OSCP mutants, and the corresponding megachannels (the electrophysiological counterpart of the PTP) are insensitive to inhibition by acidic pH in patch-clamp recordings of mitoplasts. Cells harboring the H112Q and H112Y mutations are sensitized to anoxic cell death at acidic pH. These results demonstrate that PTP channel formation and its inhibition by H+ are mediated by the F-ATP synthase.

Examining the Mechanism of Egt2 in Ergothioneine Biosynthesis

Ergothioneine (EGT), a unique histidine thiol‐derivative synthesized by certain species of bacteria and fungi, has been recognized as a powerful antioxidant that can have highly beneficial effects on human health. Ergothioneine's limited availability and potential benefits have spurred a great need to develop an efficient industrial scale production, which can best be achieved by engineering the bacterial/fungal production pathway. Interestingly, both of these pathways that produce EGT contain a terminal step in which a pyridoxal 5′‐phosphate (PLP)‐dependent C‐S lyase, termed Egt2, cleaves a carbon‐sulfoxide bond to produce EGT and pyruvate. In this study, we aimed to understand the mechanism of a novel C‐S lyase that uses sulfoxide as its substrate through a structure‐guided approach. In order to overcome a high turn‐over rate and capture the enzyme substrate complex during various stages of the reaction, we employed site‐directed mutagenesis (SDM) to mutate residues interacting with the co‐factor, PLP. Through SDM, we inserted mutations that compromised the enzyme's catalytic rate while preserving structural integrity. The crystallographic analyses revealed the structure of a highly reactive sulfenic acid intermediate that has not previously been visualized in protein chemistry.Support or Funding InformationAmerican Society of Biochemistry and Molecular Biology (ASBMB) & The University of Texas at Austin ‐ Senate of College Councils

Role of LAMP1 Binding and pH Sensing by the Spike Complex of Lassa Virus.

Lassa virus is a devastating disease-causing agent in West Africa, with a significant yearly death toll and severe long-term complications associated with its infection in survivors. In recent years, we learned that Lassa virus needs to switch receptors in a pH-dependent manner to efficiently infect cells, but neither the molecular mechanisms that allow switching nor the actual effects of switching were known. Here we investigate the activity of the viral spike complex after abrogation of its ability to switch receptors. These studies inform us about the role of switching receptors and provide new insights into how the spike senses acidic pH.

The unique histidine of F-ATP synthase subunit OSCP mediates regulation of the permeability transition by matrix pH

The “Permeability transition” (PT) is one of the most studied events that may trigger cell death and is due to a Ca2+- and ROS-dependent opening of a nonspecific pore, called PTP, whose molecular nature has been long debated. Recently, our research group has demonstrated that PTP forms from FoF1 ATP synthase dimers, demonstrating the ability of this complex to switch from the key enzyme for the aerobic synthesis of ATP into a potential cell death mediator. The goal of this PhD thesis has been to define which structural changes of ATP synthase are responsible for the pH modulation of PTP. Indeed, it is well known from nineties that the optimum matrix pH for PT occurrence is about 7.3, and a decrease leads to decreased probability of PTP opening. The pH effect has been ascribed to reversible protonation of His residues located on the PTP that can be blocked by the histidine modifying reagent diethyl pyrocarbonate (DPC). Moreover, in mammalian cells, similarly to the drug Cyclosporin A (CsA), acidic pH also promotes release from the inner membrane of the matrix protein Cyclophilin D (CyPD), which is a well-known PTP activator. As our group demonstrated that CyPD binds to the ATP Synthase OSCP subunit, mainly through electrostatic interactions and resulting in partial enzyme inhibition, the hypothesis has been advanced that the unique histidine located on OSCP, His112 according to bovine numbering, may be responsible for both the pH effects on CyPD (un)binding to ATP synthase and on PTP/ATP synthase opening. OSCPHis112 is exposed to the solvent and is located in the flexible linker region between the structured N- and C-terminal domains of OSCP. The results obtained by ATP synthase immunoprecipitation from bovine heart mitochondria showed that acidic pH induces CyPD release that is prevented by DPC, perfectly matching the effect of DPC on CyPD-PTP interaction. DPC also prevented the binding at low pH of the inhibitor protein IF1 to ATP synthase, but this effect is probably not relevant to PTP modulation. ESI-MS and ESI-MS/MS analyses of the OSCP isolated from DPC-treated mitochondria revealed that the 95-113 peptide shows a mass shift of +72 Da, which is indicative of carbethoxylation of the unique His112. These data therefore strongly support the hypothesis that OSCP His112 is part of the binding site of CyPD on the protein, so that its protonation by lowering pH favors CyPD release. Of note, this region contains several residues of glutamic acid conferring a low potential surface, which is complementary to the mainly high potential surface of CyPD. Consistently to this model, DPC inhibits the ATPase activity of ATP synthase only when CyPD is released from OSCP, i.e. in the presence of CsA and in mitochondria from CyPD-null mice. Replacement of OSCPHis112 with a Gln in HEK cells, by the CRISPR/Cas9 system, showed its involvement even in the effect of low pH on PTP opening. Indeed, the PTP open probability is not affected by acidic pH only in mutated cells, while DPC reverts the pH inhibition exclusively in wild type cells. Finally, evaluation of the structural stability of the ATP Synthase dimers at low pH by Blue-native PAGE excluded their destabilization, which could affect PTP formation. In summary, these data provide a convincing model for the pH modulation of PTP, as well as a compelling evidence that ATP synthase and PTP are the same molecular entity.

Determination of Histidine pKa Values in the Propeptides of Furin and Proprotein Convertase 1/3 Using Histidine Hydrogen-Deuterium Exchange Mass Spectrometry.

Propeptides of proprotein convertases regulate activation of their protease domains by sensing the organellar pH within the secretory pathway. Earlier experimental work highlighted the importance of a conserved histidine residue within the propeptide of a widely studied member, furin. A subsequent evolutionary analysis found an increase in histidine content within propeptides of secreted eukaryotic proteases compared with their prokaryotic orthologs. However, furin activates in the trans-golgi network at a pH of 6.5 while a paralog, proprotein convertase 1/3, activates in secretory vesicles at a pH of 5.5. It is unclear how a conserved histidine can mediate activation at two different pH values. In this manuscript, we measured the pKa values of histidines within the propeptides of furin and proprotein convertase 1/3 using a histidine hydrogen-deuterium exchange mass spectrometry approach. The high density of histidine residues combined with an abundance of basic residues provided challenges for generation of peptide ions with unique histidine residues, which were overcome by employing ETD fragmentation. During this analysis, we found slow hydrogen-deuterium exchange in residues other than histidine at basic pH. Finally, we demonstrate that the pKa of the conserved histidine in proprotein convertase 1/3 is acid-shifted compared with furin and is consistent with its lower pH of activation.

Plasticity of listeriolysin O pores and its regulation by pH and unique histidine [corrected

Pore formation of cellular membranes is an ancient mechanism of bacterial pathogenesis that allows efficient damaging of target cells. Several mechanisms have been described, however, relatively little is known about the assembly and properties of pores. Listeriolysin O (LLO) is a pH-regulated cholesterol-dependent cytolysin from the intracellular pathogen Listeria monocytogenes, which forms transmembrane β-barrel pores. Here we report that the assembly of LLO pores is rapid and efficient irrespective of pH. While pore diameters at the membrane surface are comparable at either pH 5.5 or 7.4, the distribution of pore conductances is significantly pH-dependent. This is directed by the unique residue H311, which is also important for the conformational stability of the LLO monomer and the rate of pore formation. The functional pores exhibit variations in height profiles and can reconfigure significantly by merging to other full pores or arcs. Our results indicate significant plasticity of large β-barrel pores, controlled by environmental cues like pH.

Selective interaction of Hpn-like protein with nickel, zinc and bismuth in vitro and in cells by FRET

Hpn-like (Hpnl) is a unique histidine- and glutamine-rich protein found only in Helicobacter pylori and plays a role on nickel homeostasis. We constructed the fluorescent sensor proteins CYHpnl and CYHpnl_1-48 (C-terminal glutamine-rich region truncated) using enhanced cyan and yellow fluorescent proteins (eCFP and eYFP) as the donor–acceptor pair to monitor the interactions of Hpnl with metal ions and to elucidate the role of conserved Glu-rich sequence in Hpnl by fluorescence resonance energy transfer (FRET). CYHpnl and CYHpnl_1-48 exhibited largest responses towards Ni(II) and Zn(II) over other metals studied and the binding of Bi(III) to CYHpnl was observed in the presence of an excess amount of Bi(III) ions (Kd=115±4.8μM). Moreover, both CYHpnl and CYHpnl_1-48 showed positive FRET responses towards the binding to Ni(II) and Zn(II) in Escherichia coli cells overexpressing CYHpnl and CYHpnl_1-48, whereas a decrease in FRET upon Bi(III)-binding in E. coli cells overexpressing the latter. Our study provides clear evidence on Hpnl binding to nickel in cells, and intracellular interaction of Hpnl with Bi(III) could disrupt the protein function, thus probably contributing to the efficacy of Bi(III) drugs against H. pylori.

Transposon and Deletion Mutagenesis of Genes Involved in Perchlorate Reduction in Azospira suillum PS

ABSTRACTAlthough much work on the biochemistry of the key enzymes of bacterial perchlorate reduction, chlorite dismutase, and perchlorate reductase has been published, understanding of the molecular mechanisms of this metabolism has been somewhat hampered by the lack of a clear model system amenable to genetic manipulation. Using transposon mutagenesis and clean deletions, genes important for perchlorate reduction in Azospira suillum PS have been identified both inside and outside the previously described perchlorate reduction genomic island (PRI). Transposon mutagenesis identified 18 insertions in 11 genes that completely abrogate growth via reduction of perchlorate but have no phenotype during denitrification. Of the mutants deficient in perchlorate reduction, 14 had insertions that were mapped to eight different genes within the PRI, highlighting its importance in this metabolism. To further explore the role of these genes, we also developed systems for constructing unmarked deletions and for complementing these deletions. Using these tools, every core gene in the PRI was systematically deleted; 8 of the 17 genes conserved in the PRI are essential for perchlorate respiration, including 3 genes that comprise a unique histidine kinase system. Interestingly, the other 9 genes in the PRI are not essential for perchlorate reduction and may thus have unknown functions during this metabolism. We present a model detailing our current understanding of perchlorate reduction that incorporates new concepts about this metabolism.

Probing the Protein Folding Code through the Polymer Properties of Low-Complexity Sequences

Converting primary structure into the tertiary structure of a protein remains an unsolved problem. Many proteins with highly divergent amino acid sequences have similar structures. Given the redundancy of the folding code, distinguishing signal from noise is an inherent problem. Rather than using the sequences of structurally characterized proteins to discern the code, we have adopted a strategy of first understanding the behavior of low complexity sequences using thermodynamic, kinetic and computational methods. We have inserted homopolymers of alanine, glycine and glutamine ranging from 6 to 30 residues into the N-terminus of yeast iso-1-cytochrome c. To maintain solubility, every sixth residue is lysine. Homopolymers of these amino acids are important because long repeats of these amino acids are often associated with Human disease. The homopolymers are inserted between a unique histidine and the heme allowing measurement of the thermodynamics and kinetics of His-heme loop formation under denaturing conditions ranging from 1.5 to 6 M guanidine hydrochloride (GdnHCl). Kinetic and thermodynamic loop formation data show that these homopolymers adhere closely to the expected linear dependence of loop formation probability on the logarithm of loop size predicted by the Jacobson-Stockmayer equation. Thus, the scaling exponent, ν3, and Flory's Characteristic ratio, Cn, are readily evaluated. In 6 M GdnHCl, all three hompolymers have similar ν3, but at lower GdnHCl concentrations, ν3 decreases for the Glycine-rich sequences relative to the Alanine-rich and Glutamine-rich sequences. Evaluation of Cn shows that chain stiffness follows the order: Glutamine-rich > Alanine-rich > Glycine-rich. We have also shown that insertion of aromatic amino acids into the Alanine-rich sequences significantly stabilizes His-heme loops, providing direct evidence that aromatic residues are structure-forming amino acids critical for biasing the conformational landscape of a polypeptide.

The pH sensor of the plant K+-uptake channel KAT1 is built from a sensory cloud rather than from single key amino acids

The uptake of potassium ions (K+) accompanied by an acidification of the apoplasm is a prerequisite for stomatal opening. The acidification (approximately 2-2.5 pH units) is perceived by voltage-gated inward potassium channels (K(in)) that then can open their pores with lower energy cost. The sensory units for extracellular pH in stomatal K(in) channels are proposed to be histidines exposed to the apoplasm. However, in the Arabidopsis thaliana stomatal K(in) channel KAT1, mutations in the unique histidine exposed to the solvent (His267) do not affect the pH dependency. We demonstrate in the present study that His267 of the KAT1 channel cannot sense pH changes since the neighbouring residue Phe266 shifts its pKa to undetectable values through a cation-π interaction. Instead, we show that Glu240 placed in the extracellular loop between transmembrane segments S5 and S6 is involved in the extracellular acid activation mechanism. Based on structural models we propose that this region may serve as a molecular link between the pH- and the voltage-sensor. Like Glu240, several other titratable residues could contribute to the pH-sensor of KAT1, interact with each other and even connect such residues far away from the voltage-sensor with the gating machinery of the channel.

PvAMT1;1, a Highly Selective Ammonium Transporter That Functions as H+/NH4+ Symporter

One of the main forms of nitrogen assimilated by microorganisms and plants is ammonium, despite its toxicity at low millimolar concentrations. Ammonium absorption has been demonstrated to be carried out by highly selective plasma membrane-located transporters of the AMT/MEP/Rh family and characterized by the presence of a well conserved hydrophobic pore through which ammonia is proposed to move. However, uncertainties exist regarding the exact chemical species transported by these membrane proteins, which can be in the form of either hydrophobic ammonia or charged ammonium. Here, we present the characterization of PvAMT1;1 from the common bean and demonstrate that it mediates the high affinity (micromolar), rapidly saturating (1 mM) electrogenic transport of ammonium. Activity of the transporter is enhanced by low extracellular pH, and associated with this acidic pH stimulation are changes in the reversal potential and cytoplasm acidification, indicating that PvAMT1;1 functions as an H(+)/NH(4)(+) symporter. Mutation analysis of a unique histidine present in PvAMT1;1 (H125R) leads to the stimulation of ammonium transport by decreasing the K(m) value by half and by increasing the V(max) 3-fold, without affecting the pH dependence of the symporter. In contrast, mutation of the first conserved histidine within the channel modifies the properties of PvAMT1;1, increasing its K(m) and V(max) values and transforming it into a pH-independent mechanism.

Metal Binding Ability of Cysteine-Rich Peptide Domain of ZIP13 Zn2+ Ions Transporter

The coordination modes and thermodynamic stabilities of the complexes of the cysteine-rich N-terminal domain fragment of the ZIP13 zinc transporter (MPGCPCPGCG-NH(2)) with Zn(2+), Cd(2+), Bi(3+), and Ni(2+) have been studied by potentiometric, mass spectrometric, NMR, CD, and UV-vis spectroscopic methods. All of the studied metals had similar binding modes, with the three thiol sulfurs of cysteine residues involved in metal ion coordination. The stability of the complexes formed in solution changes in the series Bi(3+) ≫ Cd(2+) > Zn(2+) > Ni(2+), the strongest being for bismuth and the weakest for nickel. The N-terminal fragment of the human metalothionein-3 (MDPETCPCP-NH(2)) and unique histidine- and cysteine-rich domain of the C-terminus of Helicobacter pyroli HspA protein (Ac-ACCHDHKKH-NH(2)) have been chosen for the comparison studies. It confirmed indirectly which groups were the anchoring ones of ZIP13 domain. Experimental data from all of the used techniques and comparisons allowed us to propose possible coordination modes for all of the studied ZIP13 complexes.

Mechanism of Heme Uptake by Heme Acquisition System A

Abstract Heme acquisition system A (HasA), secreted by various gram-negative pathogens, uses a unique histidine–iron–tyrosine (His–Fe–Tyr) coordination to scavenge heme (i.e., protoporphyrin IX–Fe complex) as an iron source. In order to study heme uptake by HasA, we utilized axial ligand mutants and protoporphyrin IX. Our results suggest that the binding of heme in apoprotein is initiated by interactions between heme and hydrophobic residues in the heme pocket. Subsequently, the sequential coordination of Tyr-75 and His-32 with the iron appears to complete holoprotein formation.

The –Cys–Cys– motif in Helicobacter pylori's Hpn and HspA proteins is an essential anchoring site for metal ions

The Hpn and HspA proteins from H. pylori are significant for nickel homeostasis and protect the cells from higher concentrations of external metal ions. Both proteins have a unique histidine- and cysteine-rich domain at the C terminus. The interactions of Ni(2+), Bi(3+), Zn(2+) and Cd(2+) ions with C-terminal Ac-CCSTSDSHHQ-NH(2) and Ac-EEGCCHGHHE-NH(2) fragments from Hpn and the Ac-GSCCHTGNHD-NH(2) sequence from HspA were studied by potentiometry, mass spectrometry, circular dichroism and UV-Vis spectroscopy. Ac-CC-NH(2) was used as a reference peptide. The studies have shown that nickel ions form planar complexes with a {2S(-),N(-)} binding mode. The thiol sulfurs of the -Cys-Cys- motif are also the anchoring sites for Bi(3+), Zn(2+) and Cd(2+) ions. The studied protein fragments have the highest affinity for Bi(3+) ions. The thermodynamic stability of Ni(2+) is much higher then that of Zn(2+).

Free radical signalling underlies inhibition of CaV3.2 T‐type calcium channels by nitrous oxide in the pain pathway

Nitrous oxide (N2O, laughing gas) has been used as an anaesthetic and analgesic for almost two centuries, but its cellular targets remain unclear. Here, we present a molecular mechanism of nitrous oxide's selective inhibition of CaV3.2 low-voltage-activated (T-type) calcium channels in pain pathways. Using site-directed mutagenesis and metal chelators such as diethylenetriamine pentaacetic acid and deferoxamine, we reveal that a unique histidine at position 191 of CaV3.2 participates in a critical metal binding site, which may in turn interact with N2O to produce reactive oxygen species (ROS). These free radicals are then likely to oxidize H191 of CaV3.2 in a localized metal-catalysed oxidation reaction. Evidence of hydrogen peroxide and free radical intermediates is given in that N2O inhibition of CaV3.2 channels is attenuated when H2O2 is neutralized by catalase. We also use the adrenochrome test as an indicator of ROS in vitro in the presence of N2O and iron. Ensuing in vivo studies indicate that mice lacking CaV3.2 channels display decreased analgesia to N2O in response to formalin-induced inflammatory pain. Furthermore, a superoxide dismutase and catalase mimetic, EUK-134, diminished pain responses to formalin in wild-type mice, but EUK-134 and N2O analgesia were not additive. This suggests that reduced ROS levels led to decreased inflammation, but without the presence of ROS, N2O was not able to provide additional analgesia. These findings reveal a novel mechanism of interaction between N2O and ion channels, furthering our understanding of this widely used analgesic in pain processing.

Propensities of Aromatic Amino Acids versus Leucine and Proline to Induce Residual Structure in the Denatured-State Ensemble of Iso-1-cytochrome c

Histidine–heme loop formation in the denatured state of a protein is a sensitive means for probing residual structure under unfolding conditions. In this study, we use a host–guest approach to investigate the relative tendencies of different amino acids to promote residual structure under denaturing conditions. The host for this work is a 6-amino-acid insert of five alanines, followed by a lysine engineered immediately following a unique histidine near the N-terminus of yeast iso-1-cytochrome c. We substitute the fourth alanine in this sequence HAAAXAK (with X = Trp, Phe, Tyr, and Leu). The effects of proline are tested with substitutions at positions 1 and 5 in the insert (HPAAAAK and HAAAAPK, respectively). Thermodynamic studies on His–heme loop formation in 3 M guanidine hydrochloride reveal significant stabilization of residual structure by aromatic amino acids, particularly Trp and Phe, and minimal stabilization of residual structure by Leu. Prolines slightly disfavor His–heme loop formation, presumably due to enhanced chain stiffness. Kinetic studies reveal that much of the change in His–heme loop stability for the aromatic amino acids is caused by a slowdown in the rate of His–heme loop breakage, indicating that residual structure is preferentially stabilized in the closed-loop form of the denatured state.

Protein stability at negative pressure

We record proton NMR spectra of the protein ubiquitin at 1 atmosphere pressure and at negative pressures (under tension), under conditions where the native and denatured states are nearly equally populated. Analysis of the unique histidine aromatic resonance of ubiquitin shows that negative pressure destabilizes the protein, in accord with a quadratic free energy dependence on pressure and temperature previously suggested in the literature. Our molecular dynamics simulations at negative pressure agree with the experimental result. In addition, molecular dynamics predicts a turnaround of the folding free energy at very low pressure. An ‘island of stability’ may exist at very negative pressures, where the protein is likely to fold into low density fluctuations of the solvent.