The binding of inhibitors to α-chymotrypsin at alkaline pH

1. The binding of the competitive inhibitor N-acetyl-d-tryptophan amide to alpha-chymotrypsin has now been studied at pH values up to 10.6, by the technique of equilibrium dialysis. 2. This binding depends on the ionization of a group on the free enzyme with apparent pK(a) 9.3 at 5 degrees . 3. This group is tentatively identified as that responsible for an enzyme conformation change at high pH values, on which the catalytic activity of the enzyme also depends.     

Biogenic Manganese Oxides Facilitate Iodide Oxidation at pH ≤ 5

Radioactive ¹²⁹I, a byproduct of nuclear power generation, can pose risks to human health if released into the environment, where its mobility is highly dependent on speciation. Based on thermodynamic principles, ¹²⁹I should exist primarily as iodide (I^?) in most terrestrial environments; however, organo-¹²⁹I and ¹²⁹iodate are also commonly detected in contaminated soils and groundwater. To investigate the capability of biogenic manganese oxides to influence iodide speciation, 17 manganese-oxidizing bacterial strains, representing six genera, were isolated from soils of the Savannah River Site, South Carolina. The isolates produced between 2.6 and 67.1 nmole Mn oxides (ml^?1 media after 25 days, pH 6.5). Results from inhibitor assays targeting extracellular enzymes and reactive oxygen species indicated that both play a role in microbe-induced Mn(II) oxidation among the strains examined. Iodide oxidation was not observed in cultures of the most active Mn-oxidizing bacteria, Chryseobacterium sp. strain SRS1 and Chromobacterium sp. strain SRS8, or the fungus, Acremonium strictum strain KR21–2. While substantial amounts of Mn(III/IV) oxides were only generated in cultures at ≥pH 6, iodide oxidation was only observed in the presence of Mn(III/IV) oxides when the pH was ≤5. Iodide oxidation was promoted to a greater extent by synthetic Mn(IV)O₂ than biogenic Mn(III/IV) oxides under these low pH conditions (≤pH 5). These results indicate that the influence of biogenic manganese oxides on iodide oxidation and immobilization is primarily limited to low pH environments.     

α-L-rhamnosidase from Aspergillus clavato-nanicus MTCC-9611 active at alkaline pH

An a-L-rhamnosidase secreting fungal strain has been isolated and identified as Aspergillus clavato-nanicus MTCC-9611. The enzyme was purified to homogeneity from the culture filtrate of the fungus using concentration by ultrafiltration membrane and ion-exchange chromatography on CM-cellulose. The native PAGE analysis confirmed the homogeneity of the purified enzyme. The SDS-PAGE analysis of the purified enzyme revealed a single protein band corresponding to the molecular weight 82 kDa. The α-L-rhamnosidase activity of Aspergillus clavato-nanicus MTCC-9611 had optimum at pH 10.0 and 50°C. The K _m values of the enzyme were 0.65 mM and 0.95 mM using p-nitrophenyl α-L-rhamnopyranoside and naringin as a substrates respectively. The enzyme transforms naringin to prunin at pH 10.0 and further hydrolysis of prunin to naringenin does not occur under these reaction conditions that makes α-L-rhamnosidase activity of Aspergillus clavato-nanicus MTCC-9611 promising enzyme to get prunin for pharmaceutical purposes.     

Introducing Titratable Water to All-Atom Molecular Dynamics at Constant?pH

Recent development of titratable coions has paved the way for realizing all-atom molecular dynamics at constant pH. To further improve physical realism, here we describe a technique in which proton titration of the solute is directly coupled to the interconversion between water and hydroxide or hydronium. We test the new method in replica-exchange continuous constant pH molecular dynamics simulations of three proteins, HP36, BBL, and HEWL. The calculated pK_a values based on 10-ns sampling per replica have the average absolute and root-mean-square errors of 0.7 and 0.9 pH units, respectively. Introducing titratable water in molecular dynamics offers a means to model proton exchange between solute and solvent, thus opening a door to gaining new insights into the intricate details of biological phenomena involving proton translocation.Solution pH is an important factor in biology. Although neutral pH in extracellular medium accounts for balanced electrostatics and proper folding of protein structures, pH gradients across cell membranes induce large conformational changes that are necessary for biological functions, such as ATP synthesis and efflux of small molecules out of the cell. To gain detailed insights into pH-dependent conformational phenomena, several constant pH molecular dynamics (pHMD) methods, based on either discrete or continuous titration coordinates, have been developed in the last decade (1–4). In the continuous pHMD (CpHMD) framework (2,4), a set of titration coordinates {λ_i} are simultaneously propagated along with the conformational degrees of freedom. Although the original CpHMD method based on the generalized Born (GB) implicit-solvent models (2,4) offers quantitative prediction of pK_a values and pH dependence of folding and conformational dynamics of proteins (5), its accuracy and applicability to highly charged systems and those with dominantly hydrophobic regions are limited due to the approximate nature of the underlying implicit-solvent models.Motivated by the above-mentioned need, three groups have made efforts to develop a CpHMD method using exclusively the explicit-solvent models (6–8). In our development, the titration of acidic and basic sites is coupled with that of coions to level the total charge of the system (8). To further improve physical realism, here we replace the coions by titratable water molecules, which not only absorb the excess charge but also enable direct modeling of solute-solvent proton exchange in classical molecular dynamics simulations.To illustrate the utility of the new methodology, we applied it to the titration simulations of three proteins that were previously used to benchmark the GB-based CpHMD. Although this work does not explore specific interactions between titratable waters and proteins, the methodology can be further tested or improved to provide a rigorous way for modeling proton transfer in molecular dynamics, which is a computationally efficient alternative to the empirical valence-bond theory-based methodologies (9,10).We define titration of water as:

• 1.Loss of a proton to give a negatively charged hydroxide,

H₂O ? OH^? + H⁺, (1)or

• 2.Gain of a proton to give a positively charged hydronium,

H₂O + H⁺ ? H₃O⁺.(2)We now couple the titration of hydroxide (Eq. 1) with that of an acidic site of the solute in the CpHMD simulation,HA+OH−⇌KaA−+H2O.(3)The use of hydronium is avoided here to prevent a potential artifact due to prolonged attraction with A⁻. Analogously, we couple the titration of hydronium (Eq. 2) with that of a basic site,BH++H2O⇌KbH3O++B.(4)Thus, effectively, a proton is transferred between the solute and solvent. However, we should note that in CpHMD simulations, titratable protons are represented by covalently attached dummies (2,4). Through varying the atomic charges and van der Waals interactions, they are seen by other atoms in the protonated state but not in the unprotonated state (see Table S1 in the Supporting Material). Furthermore, the solution proton concentration is implicitly modeled through a free energy term (2,4).In CpHMD, the reference potential of mean force (PMF) for titration is that of the model compound (blocked single amino acid in water) along λ (2,4). In the presence of cotitrating water molecules, it is necessary to add the PMF for the conversion of water to hydroxide or hydronium. One-nanosecond NPT simulations at ambient pressure and temperature were performed to calculate the average force, 〈dU/d,θ〉 at given θ-values, which are related to λ by λ = sin² θ (see Fig. S1 in the Supporting Material). Thermodynamic integration was then applied to calculate the PMF. We found that the average force can be accurately fit when assuming the PMF is quadratic in λ (Fig. 1). The same applies to the PMFs for titration of models Asp, Glu, and His. After testing on the titration of model compounds (see Table S2), we performed 10-ns all-atom CpHMD simulations with the pH replica-exchange protocol for three proteins: HP36, BBL and HEWL (see the Supporting Material for details). Most of the calculated pK_a values were converged in 10 ns per replica (see Fig. S3). Results are summarized in Fig. S4. Based on the 10-ns data, the root-mean-square (RMS) and average absolute errors are 0.9 and 0.7 pH units, respectively, while the largest absolute error is 2.5 (Glu³⁵ of HEWL). Linear regression of the calculation versus experiment gives R² of 0.8 and slope of 1.2.Open in a separate windowFigure 1Average force and potential of mean force for converting a water molecule to hydroxide (A) and hydronium. (B) (Data points) Average forces. (Dashed curves) Best fits using a linear function, 2A(λ − B). (Solid curves) Corresponding potential of mean force.

Table 1

Calculated and experimental pK_a values of three proteins

Is it possible to produce succinic acid at a low pH?

Bio-based succinate is still a matter of special emphasis in biotechnology and adjacent research areas. The vast majority of natural and engineered producers are bacterial strains that accumulate succinate under anaerobic conditions. Recently, we succeeded in obtaining an aerobic yeast strain capable of producing succinic acid at low pH. Herein, we discuss some difficulties and advantages of microbial pathways producing "succinic acid" rather than "succinate." It was concluded that the peculiar properties of the constructed yeast strain could be clarified in view of a distorted energy balance. There is evidence that in an acidic environment, the majority of the cellular energy available as ATP will be spent for proton and anion efflux. The decreased ATP:ADP ratio could essentially reduce the growth rate or even completely inhibit growth. In the same way, the preference of this elaborated strain for certain carbon sources could be explained in terms of energy balance. Nevertheless, the opportunity to exclude alkali and mineral acid waste from microbial succinate production seems environmentally friendly and cost-effective.     

Degradation of α-amylase fromAspergillus oryzae with firmly bound proteinases at pH 3.0

Incubation of highly purified -amylase fromAspergillus oryzae (EC 3.2.1.1) with 0.01M acetate buffer, pH 3.0, resulted in degradation of the -amylase. The molecular weight values of degradation products were 42 K, 37 K, and 28 K. Incubation of the purified -amylase in 0.02m phosphate buffer, pH 7.5, at 30°C for 17 h, however, resulted in no degradation of the -amylase molecule.Incubation of the purified -amylase with proangiotensin at pH 3.0 for 24 h resulted in cleavage of Tyr⁴-Ile⁵, His⁶-Pro⁷, Pro⁷-Phe⁸, Phe⁸-His⁹, and His⁹-Leu¹⁰. Thus, it appears that proteolytic activities firmly bound to -amylase are identical withAspergillus aspartic proteinase (EC 3.4.23.6) andAspergillus acid carboxypeptidase (EC 3.4.16.1).     

Solubilization of cobalt from ocean nodules at neutral pH—a novel bioprocess

A marine organism (Bacillus M1) isolated from Indian Ocean manganese nodules was characterized. The organism grew well in artificial seawater medium, at near neutral pH, 30°C and 0.25 M NaCl, and showed MnO₂-reducing activity. Growing cultures of Bacillus M1 as well as cell-free spent liquor from fully-grown cultures were employed to extract metals from the nodules. The spent liquor of cultures of the organism could dissolve around 45% cobalt (Co) at a pH of 8.2 in 2 h. Co recovery by this treatment was comparable to that in acidic leaching with 2.5 M hydrochloric acid solutions, and was independent of pulp density (w/v ratio). The amount of Co dissolved was beyond the thermodynamic solubility limit in aqueous solution at a pH of 8.2. It is inferred that the metabolites present in the spent liquor played a pivotal role in complexing the Fe (III) phase, solubilizing Co in the process. Partial characterization of spent liquor by spot tests, UV visible spectroscopy and FTIR spectroscopy, showed the presence of siderophore-like phenolic compound(s) with an attached carboxyl group that might form soluble organic complexes with Fe (III).     

Enzymatic Saccharification of Lignocelluloses Should be Conducted at Elevated pH 5.2–6.2

This study revealed that cellulose enzymatic saccharification response curves of lignocellulosic substrates were very different from those of pure cellulosic substrates in terms of optimal pH and pH operating window. The maximal enzymatic cellulose saccharification of lignocellulosic substrates occurs at substrate suspension pH 5.2–6.2, not between pH 4.8 and 5.0 as exclusively used in literature using T. reesi cellulase. Two commercial cellulase enzyme cocktails, Celluclast 1.5L and CTec2 both from Novozymes, were evaluated over a wide range of pH. The optimal ranges of measured suspension pH of 5.2–5.7 for Celluclast 1.5L and 5.5–6.2 for CTec2 were obtained using six lignocellulosic substrates produced by dilute acid, alkaline, and two sulfite pretreatments to overcome recalcitrance of lignocelluloses (SPORL) pretreatments using both a softwood and a hardwood. Furthermore, cellulose saccharification efficiency of a SPORL-pretreated lodgepole pine substrate showed a very steep increase between pH 4.7 and 5.2. Saccharification efficiency can be increased by 80 % at cellulase loading of 11.3 FPU/g glucan, i.e., from approximately 43 to 78 % simply by increasing the substrate suspension pH from 4.7 to 5.2 (buffer solution pH from 4.8 to 5.5) using Celluclast 1.5L, or by 70 % from approximately 51 to 87 % when substrate suspension pH is increased from 4.9 to 6.2 (buffer solution pH from 5.0 to 6.5) using CTec2. The enzymatic cellulose saccharification response to pH is correlated to the degree of substrate lignin sulfonation. The difference in pH-induced lignin surface charge, and therefore surface hydrophilicity and lignin–cellulase electrostatic interactions, among different substrates with different lignin content and structure is responsible for the reported different enhancements in lignocellulose saccharification at elevated pH.     

A β2-microglobulin cleavage variant fibrillates at near-physiological pH

β₂-microglobulin (β₂m) deposits as amyloid in dialysis-related amyloidosis (DRA), predominantly in joints. The molecular mechanisms underlying the amyloidogenicity of β₂m are still largely unknown. In vitro, acidic conditions, pH < 4.5, induce amyloid fibrillation of native β₂m within several days. Here, we show that amyloid fibrils are generated in less than an hour when a cleavage variant of β₂m—found in the circulation of many dialysis patients—is exposed to pH levels (pH 6.6) occurring in joints during inflammation. Aggregation and fibrillation, including seeding effects with intact, native β₂m were studied by Thioflavin T fluorescence spectroscopy, turbidimetry, capillary electrophoresis, and electron microscopy. We conclude that a biologically relevant variant of β₂m is amyloidogenic at slightly acidic pH. Also, only a very small amount of preformed fibrils of this variant is required to induce fibrillation of native β₂m. This may explain the apparent lack of detectable amounts of the variant β₂m in extracts of amyloid from DRA patients.     

The ClC-3 Cl?/H+ Antiporter Becomes Uncoupled at Low Extracellular pH

Adenovirus expressing ClC-3 (Ad-ClC-3) induces Cl⁻/H⁺ antiport current (I_ClC-3) in HEK293 cells. The outward rectification and time dependence of I_ClC-3 closely resemble an endogenous HEK293 cell acid-activated Cl⁻ current (ICl_acid) seen at extracellular pH ≤ 5.5. ICl_acid was present in smooth muscle cells from wild-type but not ClC-3 null mice. We therefore sought to determine whether these currents were related. ICl_acid was larger in cells expressing Ad-ClC-3. Protons shifted the reversal potential (E_rev) of I_ClC-3 between pH 8.2 and 6.2, but not pH 6.2 and 5.2, suggesting that Cl⁻ and H⁺ transport become uncoupled at low pH. At pH 4.0 E_rev was completely Cl⁻ dependent (55.8 ± 2.3 mV/decade). Several findings linked ClC-3 with native ICl_acid; 1) RNA interference directed at ClC-3 message reduced native ICl_acid; 2) removal of the extracellular “fast gate” (E224A) produced large currents that were pH-insensitive; and 3) wild-type I_ClC-3 and ICl_acid were both inhibited by (2-sulfonatoethyl)methanethiosulfonate (MTSES; 10–500 μm)-induced alkanethiolation at exposed cysteine residues. However, a ClC-3 mutant lacking four extracellular cysteine residues (C103_P130del) was completely resistant to MTSES. C103_P130del currents were still acid-activated, but could be distinguished from wild-type I_ClC-3 and from native ICl_acid by a much slower response to low pH. Thus, ClC-3 currents are activated by protons and ClC-3 protein may account for native ICl_acid. Low pH uncouples Cl⁻/H⁺ transport so that at pH 4.0 ClC-3 behaves as an anion-selective channel. These findings have important implications for the biology of Cl⁻/H⁺ antiporters and perhaps for pH regulation in highly acidic intracellular compartments.     

α-l-Rhamnosidase Activity in Culture Filtrate of Corticium rolfsii Enzymic Activity at Low pH

A phosphorylation system for formation of ATP from AMP by Zymolyase-treated cells of Candida boidinii (Kloeckera sp.) No. 2201 was developed as an ATP production process. This system was shown to be an energy conversion system, from a reduced C₁ -compound to ATP through reduction of NAD⁺ and oxidative phosphorylation but not substrate level phosphorylation, together with phosphorylation of AMP to ADP.

Reaction conditions for the ATP production were optimized in respect of substrate and coenzyme concentrations, pH and temperature, osmotic pressure, and oxygen supply. Under the optimal conditions, 26 mM (13 g/liter) and 8.5 dim (4g/liter) of ATP were produced with methanol and formate as C₁ -substrate, respectively.     

Bovine Carbonyl Lactoperoxidase Structure at 2.0? Resolution and Infrared Spectra as a Function of pH

Lactoperoxidase (LPO) is a hemeprotein catalyzing the oxidation of thiocyanate and I^? into antimicrobials and small aromatic organics after being itself oxidized by H₂O₂. LPO is excreted by the lungs, mammary glands, found in saliva and tears and protects mammals against bacterial, fungal and viral invasion. The Fe(II) form binds CO which inactivates LPO like many other hemeproteins. We present the 3-dimensional structure of CO?CLPO at 2.0? resolution and infrared (IR) spectra of the iron-bound CO stretch from pH?3 to 8.8?at 1 cm^?1 resolution. The observed Fe?CC?CO bond angle of 132° is more acute than the electronically related Fe(III), CN?CLPO with a Fe?CC?CN angle of 161°. The orientations of the two ligands are different with the oxygen of CO pointing towards the imidazole of distal His109 while the nitrogen of CN points away, the Fe(II) moves towards His109 while the Fe(III) moves away; both movements are consistent with a hydrogen bond between the distal His109 and CO, but not to the nitrogen of CN?CLPO. The IR spectra of CO?CLPO exhibit two major CO absorbances with pH dependent relative intensities. Both crystallographic and IR data suggest proton donation to the CO oxygen by His109 with a pK ?? 4; close to the pH of greatest enzyme turnover. The IR absorbance maxima are consistent with a first order correlation between frequency and Fe(III)/Fe(II) reduction potential at pH?7; both band widths at half-height correlate with electron density donation from Fe(II) to CO as gauged by the reduction potential.     

Crystallographic determination of reduced bovine superoxide dismutase at pH 5.0 and of anion binding to its active site

The crystal structures of dithionite-reduced bovine Cu(I),Zn superoxide dismutase and of its adducts with the inorganic anions azide and thyocyanide have been determined in a C222₁ crystal form obtained at pH?5.0. This crystal form is characterized by a high solvent content (72%) and by having the two Cu,ZnSOD monomers (A and B) in different crystal environments. One of them (B) is involved in few intermolecular crystal contacts so that it is in a more "solution like" environment, as indicated by average temperature factors which are about twice those of the other monomer. The differences in crystal packing affect the active site structures. While in the A monomer the Cu(I) is coordinated to all four histidine residues, in the B monomer the bridging His61 side chain is found disordered, implying partial detachment from copper. The same effect occurs in the structures of the anion complexes. The inorganic anions are found bound in the active site cavity, weakly interacting with copper at distances ranging from 2.5 to 2.8?Å. The copper site in the A subunit of the native enzyme structure displays significant electron density resembling a diatomic molecule, bound side-on at about 2.8?Å from the metal, which cannot be unambiguously interpreted. The crystallographic data suggest that the existence of the His61 bridge between copper and zinc is dominated by steric more than electronic factors and that the solution state favors the His61 detachment. These structures confirm the existence of an energetically available state for Cu(I) in Cu,ZnSOD where the histidinato bridge to zinc is maintained. This state appears to be favored by tighter crystal contacts. The binding of the anions in the active site cavity is different from that observed in the oxidized enzyme and it appears to be dominated by electrostatic interactions within the cavity. The anion binding mode observed may model the substrate interaction with the reduced enzyme during catalysis.     

pH值检测器

目前,测定pH值是化学实验室最常用的分析方法。然而,pH值的测定往往会因为选用的物质材料不对或保养方法不当而受到影响,下面介绍一种测定pH值的简便方法:此种pH值的测定系统由4个部分组成:一个pH值自动检测电极;一个放大器,它可以将信号译为可供仪器操作者阅读的符号;一个参考电极;以及需要测定其pH值的样品。用玻璃材料制成的检测电极实际上是一个小电池,它随着浸泡它的溶液pH值的不同而     

TheChara plasmalemma at high pH. Electrical measurements show rapid specific passive uniport of H+ or OH−

Summary Above a critical external pH (about 10.5), theChara membrane acquires new propertes. In this state the membrane potential is close to the equilibrium potentials for H⁺ and OH^–, hyperpolarizing as external pH increases with a slope of –59 mV/pH unit. The membrane conductance increases by an average factor of 2.4 above the critical pH. These changes are explained by an increase in permeability to OH^– (or H⁺). The establishment of a OH^– (or H⁺ permeable membrane at high pH suggests that the large fluxes of OH^– (or H⁺ which occur in the alkaline band in photosynthesizing cells are passive.     

Engineering a thermostable β-1,3-1,4-glucanase from Paecilomyces thermophila to improve catalytic efficiency at acidic pH

To fulfill the need for acid-tolerant and thermostable β-1,3-1,4-glucanases, an error-prone PCR and DNA-shuffling approach was employed to enhance the activity of thermostable β-1,3-1,4-glucanases from Paecilomyces thermophila (PtLic16A) at acidic pH. Mutant PtLic16AM2 was selected and characterized, and showed optimal activity at pH 5.0, corresponding to an acidic shift of 2.0 pH units relative to the wild-type enzyme. Other properties of PtLic16A such as temperature optimum and substrate specificity that are beneficial for industrial applications did not change. Based on the substituted residues of PtLic16AM2, three site-directed mutations, D56G, D221G and C263S, were designed to study these residues' roles. The amino acid residues at positions 56 and 263 were found to be important in determining optimal pH activity. Activity of the D221G variant showed no significant difference from the wild-type. Thus, it appears that the change in optimal pH for PtLic16AM2 was mainly caused by the combination of substitutions D56G and C263S. This study provides a β-1,3-1,4-glucanase (PtLic16AM2) with high potential for industrial applications.     

Regulatory Changes in the Intracellular pH and Cl– Efflux at Early Stages of Pollen Grain Germination in vitro

The regulatory role of intracellular pH changes and of transmembrane Cl^– transport in the activation of Nicotiana tabacum L. pollen grains at a stage preceding in vitro germination was studied. The acidification of the cytosol with propionic acid hindered the germination of pollen grains, whereas its alkalization by fusicoccin-stimulated H⁺-ATPase activity of plasma membranes sharply increased the germination frequency with respect to control values. The activation of pollen grains was accompanied by the Cl^– efflux. The blockage of Cl^– efflux with 1 mM ethacrynic acid significantly decreased the intracellular pH and fully inhibited germination. The results allow assumption that the intracellular pH rise and Cl^– efflux are prerequisites for pollen grain activation.     

Stabilization of pepsin on duolite for the continuous hydrolysis of bovine haemoglobin at pH2 and 40°C

Summary Several methods of pepsin immobilization have been applied in order to achieve the continuous hydrolysis of a 2.5% haemoglobin solution at pH 2 and 40°C. Methods using glutaraldehyde were unsuccesful because of the unstability of the derived enzyme at low pH. Pepsin covalently bounded to a Duolite amine resin by a carbodiimide showed a half life of 15 days during the hydrolysis of haemoglobin in a column reactor. No enzyme activity was detected in the hydrolysates. No accumulation of haem in the column was noticed which could have limited long term studies by plugging the system. Modulation of the degree of hydrolysis was also performed by changing the feeding flow rate of the reactor.     

Impact of oxidation on protein therapeutics: conformational dynamics of intact and oxidized acid-β-glucocerebrosidase at near-physiological pH

The solution dynamics of an enzyme acid-β-glucocerebrosidase (GCase) probed at a physiologically relevant (lysosomal) pH by hydrogen/deuterium exchange mass spectrometry (HDX-MS) reveals very uneven distribution of backbone amide protection across the polypeptide chain. Highly mobile segments are observed even within the catalytic cavity alongside highly protective segments, highlighting the importance of the balance between conformational stability and flexibility for enzymatic activity. Forced oxidation of GCase that resulted in a 40-60% reduction in in vitro biological activity affects the stability of some key structural elements within the catalytic site. These changes in dynamics occur on a longer time scale that is irrelevant for catalysis, effectively ruling out loss of structure in the catalytic site as a major factor contributing to the reduction of the catalytic activity. Oxidation also leads to noticeable destabilization of conformation in remote protein segments on a much larger scale, which is likely to increase the aggregation propensity of GCase and affect its bioavailability. Therefore, it appears that oxidation exerts its negative impact on the biological activity of GCase indirectly, primarily through accelerated aggregation and impaired trafficking.     

A Generic Mechanism of β2-Microglobulin Amyloid Assembly at Neutral pH Involving a Specific Proline Switch

Although numerous measurements of amyloid assembly of different proteins under distinct conditions in vitro have been performed, the molecular mechanisms underlying the specific self-association of proteins into amyloid fibrils remain obscure. Elucidating the nature of the events that initiate amyloid formation remains a particularly difficult challenge because of the heterogeneity and transient nature of the species involved. Here, we have used site-directed mutagenesis to create five proline to glycine variants in the naturally amyloidogenic protein β₂-microglobulin (β₂m). One of these variants, P5G, allowed us to isolate and characterise an intermediate containing a non-native trans Pro32 backbone conformation, a feature that is known to be required for amyloid elongation at neutral pH. By analysing oligomerisation and amyloid formation using analytical size-exclusion chromatography, multi-angle static light-scattering, analytical ultracentrifugation, circular dichroism and thioflavin T fluorescence we reveal a pathway for β₂m amyloid assembly at pH 7.5 that does not require the addition of metal ions, detergents, co-solvents or other co-factors that have been used to facilitate amyloid formation at physiological pH and temperature. Assembly is shown to involve the transient formation of a non-native monomer containing a trans P32 backbone conformation. This is followed by the formation of dimeric species and higher molecular mass oligomers that accumulate before the development of amyloid fibrils. On the basis of these results, we propose a generic mechanism for β₂m fibrillogenesis at neutral pH that is consistent with the wide range of published studies of this protein. In this mechanism, amyloid formation is initiated by a specific cis to trans proline switch, the rate of which we show to be controlled by the amino acid sequence proximal to P32 and to the applied solution conditions.