The Human α-Lactalbumin Molten Globule: Comparison of Structural Preferences at pH 2 and pH 7

Structural investigations of molten globules provide an important contribution towards understanding protein folding pathways. A close similarity between equilibrium molten globule states and kinetic species observed during refolding has been reported for several proteins. However, the experimental conditions, and in particular the pH, under which the equilibrium and kinetic species are studied often differ significantly. For human α-lactalbumin (α-LA), the equilibrium molten globule is most often studied at pH 2, the so-called A-state, while kinetic refolding experiments are performed at neutral pH. α-LA contains a large number of acidic amino acid residues that may influence the properties of the molten globule differently at low and neutral pH. In this study, we investigate the structural preferences of the α-LA molten globule at pH 7 at the level of individual residues using nuclear magnetic resonance spectroscopy and compare these data with previous results obtained at pH 2. We show that differences exist in the conformational ensemble that describes the α-LA molten globule at these two pH values. The molten globule at pH 7 is generally less stable than that at the low pH A-state. Most notable are differences in the stability of structure for the C-helix and the calcium-binding loop that precedes it and differences in the contribution of long-range hydrophobic contacts between the N-terminal and C-terminal regions of the α-domain to the stability of the molten globule. Our results are discussed in the context of previous studies of the α-LA molten globule and can be used to reconcile apparent discrepancies in published data relating to the C-helix. In the light of our results, the low pH A-state may not be the best model for the kinetic molten globule observed during refolding of α-LA. The pH-dependent effects reported here for α-LA may be of relevance in comparisons of equilibrium and kinetic molten globules of other proteins.     

The “Pre-Molten Globule,” a New Intermediate in Protein Folding

In vitro folding studies of several proteins revealed the formation, within 2–4 msec, of transient intermediates with a large far-UV ellipticity but no amide proton protection. To solve the contradiction between the secondary structure contents estimated by these two methods, we characterized the isolated C-terminal fragment F2 of the tryptophan synthase β₂ subunit. In β₂, F2 forms its tertiary interactions with the F1 N-terminal region. Hence, in the absence of F1, isolated F2 should remain at an early folding stage with no long-range interactions. We shall show that isolated F2 folds into, and remains in, a “state” called the pre-molten globule, that indeed corresponds to a 2- to 4-msec intermediate. This condensed, but not compact, “state” corresponds to an array of conformations in rapid equilibrium comprising native as well as nonnative secondary structures. It fits the “new view” on the folding process.     

Illuminating the Off-Pathway Nature of the Molten Globule Folding Intermediate of an α-β Parallel Protein

Partially folded protein species transiently form during folding of most proteins. Often, these species are molten globules, which may be on- or off-pathway to the native state. Molten globules are ensembles of interconverting protein conformers that have a substantial amount of secondary structure, but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to solvent-exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule observed during folding of the 179-residue apoflavodoxin from Azotobacter vinelandii is off-pathway, as it has to unfold before native protein can form. Here, we study folding of apoflavodoxin and characterize its molten globule using fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET). Apoflavodoxin is site-specifically labeled with fluorescent donor and acceptor dyes, utilizing dye-inaccessibility of Cys69 in cofactor-bound protein. Donor (i.e., Alexa Fluor 488) is covalently attached to Cys69 in all apoflavodoxin variants used. Acceptor (i.e., Alexa Fluor 568) is coupled to Cys1, Cys131 and Cys178, respectively. Our FRET data show that apoflavodoxin’s molten globule forms in a non-cooperative manner and that its N-terminal 69 residues fold last. In addition, striking conformational differences between molten globule and native protein are revealed, because the inter-label distances sampled in the 111-residue C-terminal segment of the molten globule are shorter than observed for native apoflavodoxin. Thus, FRET sheds light on the off-pathway nature of the molten globule during folding of an α-β parallel protein.     

Role for β2-Microglobulin in Echovirus Infection of Rhabdomyosarcoma Cells

A monoclonal antibody (MAb) that blocks most echoviruses (EVs) from infecting rhabdomyosarcoma (RD) cells has been isolated. By using the CELICS cloning method (T. Ward, P. A. Pipkin, N. A. Clarkson, D. M. Stone, P. D. Minor, and J. W. Almond, EMBO J. 13:5070–5074, 1994), the ligand for this antibody has been identified as β2-microglobulin (β2m), the 12-kDa protein that associates with class I heavy chains to form class I HLA complexes. A commercial MAb (MAb 1350) against β2m was also found to block EV7 infection without affecting binding to its receptor, DAF, or replication of EV7 viral RNA inside cells. Entry of EV7 into cells was reduced by only 30% by antibody and cytochalasin D, an inhibitor of endocytosis mediated by caveolae and clathrin-coated pits, but was not significantly reduced by sodium azide. The block to virus entry by cytochalasin D was additive to the block induced by antibody. We suggest that EV7 rapidly enters into a multicomponent receptor complex prior to entry into cells and that this initial entry event requires β2m or class I HLA for infection to proceed.Echoviruses (EVs) are members of the Enterovirus genus of the family Picornaviridae and are important human pathogens. They are associated with a wide spectrum of clinical syndromes, including rashes, diarrhea, aseptic meningitis, respiratory disease, and possibly conditions such as chronic fatigue syndrome. This range of clinical manifestations is probably a reflection of virus tissue tropisms, which seem to be mediated, at least in part, by utilization of a range of cellular receptors.Anti-cell surface monoclonal antibodies (MAbs) that block EV infection have been isolated previously and have been used to determine the identity of some of these receptors. In 1992 Bergelson et al. demonstrated that EV serotypes 1 and 8 use the collagen receptor VLA-2 (6) by attaching to the α2 subunit (7). Previously, we and others have shown that a regulator of complement activity, decay-accelerating factor (DAF), is the receptor for a range of hemagglutinating EVs (3, 37). Other EVs appear to use neither of these, but the identity of their receptor(s) is unknown. Mbida et al. have isolated a MAb (MAb 143) that blocks most EV serotypes from infecting a range of cell types. MAb 143 was also found to block coxsackievirus A9 but not poliovirus or coxsackievirus serotypes B1 to B6 (21). The ligand for MAb 143 was found by affinity purification to be an unknown 44-kDa glycoprotein (22). It was therefore suggested that the 44-kDa protein was part of a multicomponent receptor complex used by most EVs to infect cells. A direct role for the 44-kDa protein in virus attachment seems unlikely, since MAb 143 blocks infection by the viruses that have been shown to use other proteins, such as DAF (3, 37) and VLA-2 (6), as their primary receptors.Here, we report the isolation of a MAb similar to that described by Mbida et al. (21, 22) and describe the cloning and identification of its ligand. The ligand is β2-microglobulin (β2m), a 12-kDa protein that associates with the class I HLA heavy chains (44 kDa) and presents antigenic peptides (20). We show that MAbs to β2m block EV infection partly by reducing the entry of virus into cells, although other postbinding effects cannot be ruled out.     

Native-State Heterogeneity of β2-Microglobulin as Revealed by Kinetic Folding and Real-Time NMR Experiments

The kinetic folding of β₂-microglobulin from the acid-denatured state was investigated by interrupted-unfolding and interrupted-refolding experiments using stopped-flow double-jump techniques. In the interrupted unfolding, we first unfolded the protein by a pH jump from pH 7.5 to pH 2.0, and the kinetic refolding assay was carried out by the reverse pH jump by monitoring tryptophan fluorescence. Similarly, in the interrupted refolding, we first refolded the protein by a pH jump from pH 2.0 to pH 7.5 and used a guanidine hydrochloride (GdnHCl) concentration jump as well as the reverse pH jump as unfolding assays. Based on these experiments, the folding is represented by a parallel-pathway model, in which the molecule with the correct Pro32 cis isomer refolds rapidly with a rate constant of 5–6 s^? 1, while the molecule with the Pro32 trans isomer refolds more slowly (pH 7.5 and 25 °C). At the last step of folding, the native-like trans conformer produced on the latter pathway isomerizes very slowly (0.001–0.002 s^? 1) into the native cis conformer. In the GdnHCl-induced unfolding assays in the interrupted refolding, the native-like trans conformer unfolded remarkably faster than the native cis conformer, and the direct GdnHCl-induced unfolding was also biphasic, indicating that the native-like trans conformer is populated at a significant level under the native condition. The one-dimensional NMR and the real-time NMR experiments of refolding further indicated that the population of the trans conformer increases up to 7–9% under a more physiological condition (pH 7.5 and 37 °C).     

Heterogeneity of β2-Microglobulin in Human Urine

Purification and characterization of β₂-microglobulin from human urine was performed. The yield was 30.1%, and 150.4 mg of β₂-microglobulin was obtained. The final preparation of β₂-microglobulin obtained showed three bands on disc gel electrophoresis at pH 9.5, and all of them have immunological activity. However, these three bands migrated as a single band on disc gel electrophoresis at pH 4.3. It is concluded that the three bands observed on disc gel electrophoresis at pH 9.5 were charge isomers. The isoelectric points of isomers were determined by isotachophoresis and two of them were 5.4 and 5.9 respectively, while the other one was not determined.     

Oligomeric States along the Folding Pathways of β2-Microglobulin: Kinetics,Thermodynamics, and Structure

The transition of proteins from their soluble functional state to amyloid fibrils and aggregates is associated with the onset of several human diseases. Protein aggregation often requires some structural reshaping and the subsequent formation of intermolecular contacts. Therefore, the study of the conformation of excited protein states and their ability to form oligomers is of primary importance for understanding the molecular basis of amyloid fibril formation. Here, we investigated the oligomerization processes that occur along the folding of the amyloidogenic human protein β2-microglobulin. The combination of real-time two-dimensional NMR data with real-time small-angle X-ray scattering measurements allowed us to derive thermodynamic and kinetic information on protein oligomerization of different conformational states populated along the folding pathways. In particular, we could demonstrate that a long-lived folding intermediate (I-state) has a higher propensity to oligomerize compared to the native state. Our data agree well with a simple five-state kinetic model that involves only monomeric and dimeric species. The dimers have an elongated shape with the dimerization interface located at the apical side of β2-microglobulin close to Pro32, the residue that has a trans conformation in the I-state and a cis conformation in the native (N) state. Our experimental data suggest that partial unfolding in the apical half of the protein close to Pro32 leads to an excited state conformation with enhanced propensity for oligomerization. This excited state becomes more populated in the transient I-state due to the destabilization of the native conformation by the trans-Pro32 configuration.     

The Role of β-Hydroxypropionate in Ethylene Biosynthesis

To search precursors of ethylene in banana fruits, ethylene formation from acetate-2-¹⁴C and fumarate-2,3-¹⁴C by banana slices was studied. Ethylene-¹⁴C formation from acetate-2-^l4C was reduced by the addition of malonate or β-hydroxypropionate, and it was enhanced in a sealed chamber in comparison with the case in an aeration chamber. No label of fumarate-2,3-¹⁴C was incorporated into ethylene.

From these facts it was suggested that acetate-2-¹⁴C was incorporated into ethylene via malonate and β-hydroxypropionate. Participation of fumarate in ethylene biosynthesis of banana fruits was ruled out. β-Hydroxypropionate was postulated as an effective precursor of ethylene formation from acetate-2-^l4C.     

Residue-Specific Analysis of Frustration in the Folding Landscape of Repeat β/α Protein Apoflavodoxin

Flavodoxin adopts the common repeat β/α topology and folds in a complex kinetic reaction with intermediates. To better understand this reaction, we analyzed a set of Desulfovibrio desulfuricans apoflavodoxin variants with point mutations in most secondary structure elements by in vitro and in silico methods. By equilibrium unfolding experiments, we first revealed how different secondary structure elements contribute to overall protein resistance to heat and urea. Next, using stopped-flow mixing coupled with far-UV circular dichroism, we probed how individual residues affect the amount of structure formed in the experimentally detected burst-phase intermediate. Together with in silico folding route analysis of the same point-mutated variants and computation of growth in nucleation size during early folding, computer simulations suggested the presence of two competing folding nuclei at opposite sides of the central β-strand 3 (i.e., at β-strands 1 and 4), which cause early topological frustration (i.e., misfolding) in the folding landscape. Particularly, the extent of heterogeneity in folding nuclei growth correlates with the in vitro burst-phase circular dichroism amplitude. In addition, ?-value analysis (in vitro and in silico) of the overall folding barrier to apoflavodoxin's native state revealed that native-like interactions in most of the β-strands must form in transition state. Our study reveals that an imbalanced competition between the two sides of apoflavodoxin's central β-sheet directs initial misfolding, while proper alignment on both sides of β-strand 3 is necessary for productive folding.     

Formation,Stability and In Vitro Digestion of β-carotene in Oil-in-Water Milk Fat Globule Membrane Protein Emulsions

The formation, stability and in vitro digestion of milk fat globule membrane (MFGM) proteins stabilized emulsions with 0.2 wt% β-carotene were investigated. The average particle size of β-carotene emulsions stabilized with various MFGM proteins levels (1%, 2%, 3%, 4%, 5% wt%) decreased with the increase of MFGM proteins levels. When MFGM proteins concentration in emulsions is above 2%, the average particle size of β-carotene emulsions is below 1.0 μm. A quite stable emulsion was formed at pH 6.0 and 7.0, but particle size increased with decrease in acidity of the β-carotene emulsion. β-carotene emulsions stabilized with MFGM proteins were stable with a certain salt concentrations (0–500 mMNaCl). β-carotene emulsions were quite stable to aggregation of the particles at elevated temperature and time (85 °C for 90 min). At the same time, β-carotene emulsions were stable against degradation under heat treatment conditions. In vitro digestion of β-carotene emulsion showed the mean particle size of β-carotene emulsions stabilized with MFGM proteins in the simulated stomach conditions and intestinal conditions is larger than that of initial emulsions and simulated mouth conditions. Confocal laser scanning microscopy of β-carotene MFGM proteins emulsions also showed the corresponding results to different vitro digestion model. There was a rapid release of free fatty acid (FFA) during the first 10 min and after this period, an almost constant 70% digestion extent was reached. Approximately 80% of β-carotene was released within 2 h of incubation under the simulated intestinal fluid. These results showed that MFGM protein can be used as a good emulsifier in emulsion stabilization, β-carotene rapid release as well as lipophilic bioactive compounds delivery.     

Novel Antibacterial Activity of β2-Microglobulin in Human Amniotic Fluid

An antibacterial protein (about 12 kDa) was isolated from human amniotic fluid through dialysis, ultrafiltration and C18 reversed-phase HPLC steps. Automated Edman degradation showed that the N-terminal sequence of the antibacterial protein was NH₂-Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Tyr-Ser-Arg-His-Pro-Ala-Glu-Asn-Gly-. The N-terminal sequence of the antibacterial protein was found to be identical to that of β₂-microglobulin, a component of MHC class I molecules, which are present on all nucleated cells. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) revealed that the molecular mass of the antibacterial protein was 11,631 Da. This antibacterial protein, β₂M, possessed potent antibacterial activity against pathogenic bacteria. Specially, antibacterial activity was observed in potassium buffer, and potassium ion was found to be critical for the antibacterial activity. Interestingly, the antibacterial action of β₂M was associated with dissipation of the transmembrane potential, but the protein did not cause damage to the membrane that would result in SYTOX green uptake. In addition, stimulation of WISH amniotic epithelial cells with the bacterial endotoxin lipopolysaccharide (LPS) induced dose-dependent upregulation of β₂M mRNA expression. These results suggest that β₂M contributes to a self-defense response when amniotic cells are exposed to pathogens.     

A Non-Native α-Helix Is Formed in the β-Sheet Region of the Molten Globule State of Canine Milk Lysozyme

The native and the molten globule states (N and MG states, respectively) of canine milk lysozyme (CML) were examined by CD spectroscopy and AGADIR algorithm, a helix-coil transition program. It revealed that the helical content of the MG state was higher than that of the N-state, suggesting that non-native alpha-helix is formed in the MG state of CML. Using AGADIR, it indicated the possibility of alpha-helix formation in the third beta-strand region in the MG state. To investigate this possibility, we designed a mutant, Q58P, in which the helical propensity of the MG state was significantly decreased around the third beta-strand region. It appeared that the absolute ellipticity value at 222 nm of the mutant in the MG state was smaller than that of the wild-type protein. It could be assumed that the non-native alpha-helix is formed around the third beta-strand region of wild-type CML in the MG state.     

Native-unlike Long-lived Intermediates along the Folding Pathway of the Amyloidogenic Protein ��2-Microglobulin Revealed by Real-time Two-dimensional NMR

β2-microglobulin (β2m), the light chain of class I major histocompatibility complex, is responsible for the dialysis-related amyloidosis and, in patients undergoing long term dialysis, the full-length and chemically unmodified β2m converts into amyloid fibrils. The protein, belonging to the immunoglobulin superfamily, in common to other members of this family, experiences during its folding a long-lived intermediate associated to the trans-to-cis isomerization of Pro-32 that has been addressed as the precursor of the amyloid fibril formation. In this respect, previous studies on the W60G β2m mutant, showing that the lack of Trp-60 prevents fibril formation in mild aggregating condition, prompted us to reinvestigate the refolding kinetics of wild type and W60G β2m at atomic resolution by real-time NMR. The analysis, conducted at ambient temperature by the band selective flip angle short transient real-time two-dimensional NMR techniques and probing the β2m states every 15 s, revealed a more complex folding energy landscape than previously reported for wild type β2m, involving more than a single intermediate species, and shedding new light into the fibrillogenic pathway. Moreover, a significant difference in the kinetic scheme previously characterized by optical spectroscopic methods was discovered for the W60G β2m mutant.     

The Role of Dbf4-Dependent Protein Kinase in DNA Polymerase ζ-Dependent Mutagenesis in Saccharomyces cerevisiae

The yeast Dbf4-dependent kinase (DDK) (composed of Dbf4 and Cdc7 subunits) is an essential, conserved Ser/Thr protein kinase that regulates multiple processes in the cell, including DNA replication, recombination and induced mutagenesis. Only DDK substrates important for replication and recombination have been identified. Consequently, the mechanism by which DDK regulates mutagenesis is unknown. The yeast mcm5-bob1 mutation that bypasses DDK’s essential role in DNA replication was used here to examine whether loss of DDK affects spontaneous as well as induced mutagenesis. Using the sensitive lys2ΔA746 frameshift reversion assay, we show DDK is required to generate “complex” spontaneous mutations, which are a hallmark of the Polζ translesion synthesis DNA polymerase. DDK co-immunoprecipitated with the Rev7 regulatory, but not with the Rev3 polymerase subunit of Polζ. Conversely, Rev7 bound mainly to the Cdc7 kinase subunit and not to Dbf4. The Rev7 subunit of Polζ may be regulated by DDK phosphorylation as immunoprecipitates of yeast Cdc7 and also recombinant Xenopus DDK phosphorylated GST-Rev7 in vitro. In addition to promoting Polζ-dependent mutagenesis, DDK was also important for generating Polζ-independent large deletions that revert the lys2ΔA746 allele. The decrease in large deletions observed in the absence of DDK likely results from an increase in the rate of replication fork restart after an encounter with spontaneous DNA damage. Finally, nonepistatic, additive/synergistic UV sensitivity was observed in cdc7Δ pol32Δ and cdc7Δ pol30-K127R,K164R double mutants, suggesting that DDK may regulate Rev7 protein during postreplication “gap filling” rather than during “polymerase switching” by ubiquitinated and sumoylated modified Pol30 (PCNA) and Pol32.     

Photorespiration: Its Role in the Productive Process and Evolution of С<Subscript>4</Subscript> Plants

The review surveys the modern concepts of photorespiration, its role in the productive process, redox-regulation, and integration of energy-transforming processes of the photosynthesizing cell and, also, in evolution of C₄ plants. Special attention is given to methods to explore photorespiration. Principal strategies that are aimed at improvements in photosynthesis intensity and in plant growth by manipulations with photorespiration based on up-to-date technologies are considered.     

Role of AKAP79/150 Protein in β1-Adrenergic Receptor Trafficking and Signaling in Mammalian Cells

Protein kinase A-anchoring proteins (AKAPs) participate in the formation of macromolecular signaling complexes that include protein kinases, ion channels, effector enzymes, and G-protein-coupled receptors. We examined the role of AKAP79/150 (AKAP5) in trafficking and signaling of the β₁-adrenergic receptor (β₁-AR). shRNA-mediated down-regulation of AKAP5 in HEK-293 cells inhibited the recycling of the β₁-AR. Recycling of the β₁-AR in AKAP5 knockdown cells was rescued by shRNA-resistant AKAP5. However, truncated mutants of AKAP5 with deletions in the domains involved in membrane targeting or in binding to calcineurin or PKA failed to restore the recycling of the β₁-AR, indicating that full-length AKAP5 was required. Furthermore, recycling of the β₁-AR in rat neonatal cardiac myocytes was dependent on targeting the AKAP5-PKA complex to the C-terminal tail of the β₁-AR. To analyze the role of AKAP5 more directly, recycling of the β₁-AR was determined in ventricular myocytes from AKAP5^−/− mice. In AKAP5^−/− myocytes, the agonist-internalized β₁-AR did not recycle, except when full-length AKAP5 was reintroduced. These data indicate that AKAP5 exerted specific and profound effects on β₁-AR recycling in mammalian cells. Biochemical or real time FRET-based imaging of cyclic AMP revealed that deletion of AKAP5 sensitized the cardiac β₁-AR signaling pathway to isoproterenol. Moreover, isoproterenol-mediated increase in contraction rate, surface area, or expression of β-myosin heavy chains was significantly greater in AKAP5^−/− myocytes than in AKAP5^+/+ myocytes. These results indicate a significant role for the AKAP5 scaffold in signaling and trafficking of the β₁-AR in cardiac myocytes and mammalian cells.     

The Inhibitory Role of Lactacystin and β-Lactacystinon T-cell Activation and Proliferation

To evaluate the effects of proteasome inhibitors lactacystin (LAC) and beta-lactacystin (beta-LAC) on the proliferation and activation of T lymphocytes, flow cytometry was used to analyze the proliferation and the expression of CD69, CD25 and CD3 of T lymphocytes activated by PHA. Furthermore, the expressions of PA28 and IL-2 mRNA were assayed by competitive RT-PCR. The results indicated that: (1) LAC and beta-LAC significantly decreased the incorporation of BrdU and inhibited T lymphocytes proliferation in T lymphocytes activated by PHA; (2) although LAC and beta-LAC did not affect the expression of CD69 at any time, they significantly inhibited the expression of CD25 (48 h, 72 h, P<0.05); (3) in comparison with control, LAC and beta--LAC significantly down-regulated the expression of PA28 and IL-2 mRNA (48 h, 72 h, P<0.05). LAC and beta-LAC significantly inhibited the proliferation and activation of T cells. Mechanisms involved are inhibition of CD25 and down-regulation of PA28 and IL-2 mRNA expressions.     

Role of hydrophobic interactions and salt-bridges in β-hairpin folding

β-Hairpins are the simplest form of β-sheets which, due to the presence of long-range interactions, can be considered as tertiary structures. Molecular dynamics simulation is a powerful tool that can unravel whole pathways of protein folding/unfolding at atomic resolution. We have performed several molecular dynamics simulations, to a total of over 250 ns, of a β-hairpin peptide in water using GROMACS. We show that hydrophobic interactions are necessary for initiating the folding of the peptide. Once formed, the peptide is stabilized by hydrogen bonds and disruption of hydrophobic interactions in the folded peptide does not denature the structure. In the absence of hydrophobic interactions, the peptide fails to fold. However, the introduction of a salt-bridge compensates for the loss of hydrophobic interactions to a certain extent. Figure Model of b-hairpin folding: Folding is initiated by hydrophobic interactions (Brown circles). The folded structure, once formed, is stabilized by hydrogen bonds (red lines) and is unaffected by loss of hydrophobic contacts