Biogenesis of the chloroplast phosphate translocator

Calcium-dependent proteolysis of several polypeptides from rat brain and synaptosomal cytosol was observed including proteolysis of polypeptides of M_r 340 000 and 300 000. These latter polypeptides comigrated with high-M_r microtubule-associated proteins of microtubule preparations from brain or synaptosomal cytosol. Calcium influx into intact synaptosomes due to depolarisation with high potassium or veratridine or treatment with the ionophore A23187 did not result in Ca²⁺-dependent proteolysis of any polypeptides. This may be due to the low calcium sensitivity of the protease since no proteolysis of the M_r 340 000 and 300 000 polypeptides was seen in synaptosomal cytosal at < 10 μM free Ca²⁺.     

Detection of Sendai virus fusion with human erythrocytes by fluorescence photobleaching recovery

The subunit stoichiometry of the ATP synthetase (CF₁-CF₀) immunoprecipitated from Triton X-100 extracts of chloroplast thylakoid membranes was determined to be α₃, β₃, γ, δ, ? (CF₁) and I_0.3, II_0.6–0.9, III₄₍₆₎ (CF₀). Antibodies against the polypeptides α, β, γ, δ, I, II and ? combined specifically with the isolated subunits as analysed by the protein blotting method. Applying this technique, antibodies against the CF₁ subunits were found to form complexes with the corresponding polypeptides of thylakoids, whereas those against I (M_r 20 000) and II (M_r 17 000) combined with M_r 26 000 and M_r 24 500 membrane polypeptides, respectively. The M_r 26 000 polypeptide was identified as the major subunits of the light-harvesting chlorophyll a/b-protein (LHCP) complex and the M_r 24 500 component seems to be functionally connected with this complex. From the results it is concluded that the chloroplast ATP synthetase consists of the subunit of the α, β, γ, δ, ? and III (proteolipid only and that proteolytically altered LHCP polypeptides bind artifically to the protein complex during isolation.     

Polypeptide composition and biosynthesis of the yolk proteins in the firebrat,Thermobia domestica (insecta,thysanura)

Ion-exchange chromatography of crude ovarian extracts of the primitive insect Thermobia domestica allowed the separation, in native conditions, of major and minor vitellins of molecular weights of 300,000 and 430,000, respectively. Their polypeptide subunits were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunotransfer using an antiserum prepared against major vitellin. This protein was resolved into large (M_r 166,000–212,000) and small (around M_r 50,000) polypeptides. Minor vitellin, on the other hand, exclusively contained small polypeptides that are immunologically different from those of the major vitellin. Vitellogenin polypeptides from the hemolymph of mature females exhibited electrophoretic mobilities and immunological properties similar to vitellin polypeptides. Pulse-chase experiments showed that the female fat body synthesizes radioactive and immunoprecipitable proteins, whose polypeptide pattern is close to that of the major vitellogenin. However, part of the primary vitellogenic polypeptides, at M_r 210,000 and 212,000, is rapidly processed to M_r 176,000 and 182,000 subunits. These two polypeptides, as well as the precursors, enter into the composition of the major hemolymph vitellogenin. Finally, processing of the still uncleaved 210,000–212,000 polypeptides takes place in the ovary, which performs the same step of vitellogenin maturation as the fat body.     

Leishmania tropica: Surface antigens of intracellular and flagellate forms

Promastigotes and amastigotes of Leishmania tropica were surface-radioiodinated using the lactoperoxidase technique. Detergent lysates of the labeled organisms were analyzed by two-dimensional gel electrophoresis. Analysis of radioiodinated promastigote membrane proteins revealed six major and some minor acidic polypeptides. Analysis of the amastigote membrane proteins revealed six major proteins, mostly acidic, and some poorly resolved basic proteins. Four of the major membrane proteins appeared to be common to the two parasitic forms (M_r 67,000, M_r 50,000, M_r 68,000, and M_r 80,000). These polypeptides were recognized by antipromastigote antibodies as well as antibodies from CBA/H mice that had recovered from infection. Peptide mapping confirmed their homology in the two parasite forms. One polypeptide appeared to be specific for the promastigote (M_r 50,000) and two polypeptides appeared to be specific for the amastigote form of the parasite (M_r 94,000 and M_r 43,000).     

In Vitro Processing of Precursors of Thylakoid Membrane Proteins of Chlamydomonas reinhardtii y-1

Studies of in vitro processing of precursors of the major chlorophyll a/b-binding polypeptides of Chlamydomonas reinhardtii y-1 were undertaken to define the precursor-product relationships. Analysis of translates, prepared from C. reinhardtii poly(A)-rich RNA in a rabbit reticulocyte lysate system, which were incubated with the soluble fraction from C. reinhardtii cells, showed that the 31,500 relative molecular mass (M_r) precursor was converted to the M_r 29,500 thylakoid membrane polypeptide whereas the M_r 30,000 precursor was converted to the M_r 26,000 product. Furthermore, the M_r 31,500 polypeptide, when bound to antibodies, was not processed to the mature polypeptide of M_r 29,500, although the presence of antibodies did not prevent the precursor of M_r 30,000 from being converted to the mature M_r 26,000 polypeptide. The mature fraction of M_r 26,000, was separated into two bands corresponding to polypeptides 16 and 17 in the electrophoretic system of Chua and Bennoun (1975 Proc Natl Acad Sci USA 72: 2175-2179).

Processing activity was present in the soluble fraction obtained from cells grown in the light or in the dark. Therefore, processing of the precursor polypeptides does not appear to be involved in the regulation by light of the accumulation of these polypeptides in thylakoid membranes.

     

ATP-synthetase complex (F1F0) from Escherichia coli. Purification and characterization of subunits A and B of the F0 part

An intact B890 light-harvesting pigment—protein complex has been obtained from Rhodospirillum rubrum strain S1. We show that this complex contains two types of low-M_r polypeptide. Both these polypeptides are present in the intact chromatophore membrane. Analysis of the pigment content of this complex suggests that per pair of polypeptides the complex contains 2 molecules of bacteriochlorophyll and one molecule of carotenoid.     

Complex formation between metarhodopsin II and GTP-binding protein in bovine photoreceptor membranes leads to a shift of the photoproduct equilibrium

Cap binding protein (CBP)-related polypeptides were identified in different cytoplasmic RNP particles of embryonic chick muscles using monoclonal antibody to purified CBP. A single immunoreactive peptide (M_r 78000) was present in preparations of both free mRNP particles and a novel 10 S translation inhibitory RNP particle. In contrast, proteins isolated from these particles showed two new low-M_r immunoreactive peptides (M_r 43000 and M_r 29000). No CBP related protein could be detected in polysomal mRNP, although an immunoreactive M_r 43000 CBP-related protein was present in polysomes. The relevance of the association of different CBP-related polypeptides with cytoplasmic RNP particles and polysomes are discussed.     

Structural polypeptides and products of late genes of bacteriophage Mu: Characterization and functional aspects

This paper describes the identification and functional role of late gene products of bacteriophage Mu, including an analysis of the structural proteins of the Mu virion.In vitro reconstitution of infectious phage particles has shown that four genes (E, D, I, J) control the formation of phage heads and that a cluster of eight genes (K, L, M, N, P, Q, R, S) controls the formation of phage tails.Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of Mu polypeptides synthesized in Escherichia coli minicells infected by Mu phages carrying amber mutations in various late genes has resulted in the identification of the products of gene C (15.5 × 10³M_r); H (64 × 10³M_r); F (54 × 10³M_r); G (16 × 10³M_r); L (55 × 10³M_r); N (60 × 10³M_r); P (43 × 10³M_r) and S (56 × 10³M_r). Minicells infected with λpMu hybrid phages and deletion mutants of Mu were used to identify polypeptides encoded by the V-β region of the Mu genome. These are the products of genes V, W or R (41.5 × 10³M_r, and 45 × 10³M_r); U (20.5 × 10³M_r) and of genes located in the β region (24 × 10³M_r (gpgin) and 37 × 10³M_r (possibly gpmom)).Analytical separation of the proteins of the Mu virion revealed that it consists of a major head polypeptide with a molecular weight of 33 × 10³, a second head polypeptide of 54 × 10³ (gpF) and two major tail polypeptides with molecular weights of 55 × 10³ and 12.5 × 10³ (gpL and gpY, respectively). In addition, there are five minor components of the tail (including gpN, gpS and gpU) and approximately seven minor components of the head structure of the virion (including gpH).     

Immunolocalization of the 110,000 molecular weight cytoskeletal protein of intestinal microvilli

The core structures of microvilli from absorptive cells of the intestinal epithelium are primarily composed of calmodulin (M_r 16,000), actin (M_r 43,000), villin (M_r 95,000) and a protein of M_r 110,000. We have isolated this protein and raised antibodies against it. The antibodies interact specifically with villin and M_r 110,000 polypeptides present in isolated microvilli or brush borders. However, after absorption on an immobilized villin preparation, these antibodies still immunoprecipitate the M_r 110,000 protein but not villin. Thus, these two proteins appear to share some antigenic determinants but also contain other determinants specific for each protein. Immunolocalization studies have been performed using specific antibodies against the M_r 110,000 protein. Immunofluorescent studies on thin frozen sections of intestinal cells show that this protein is located in the brush border and at the basolateral faces of these polarized cells. Immunoferritin studies on rat brush borders demembranated with the detergent Triton X-100 show the association of the M_r 110,000 protein with core filaments of microvilli, as well as with some filaments localized in the terminal web network.Using sealed, right-side-out vesicles prepared from pig intestinal mucosa in the presence of Ca²⁺ and Mg²⁺, a polypeptide of M_r 140,000 was found to be a major component of the Triton X-100 insoluble pellet. This protein is a minor component of an equivalent pellet obtained from isolated microvilli prepared in the presence of EDTA. The significance of this M_r 140,000 polypeptide associated with the core residue of intestinal microvilli is discussed.     

Cell type-specific differences in protein composition of nuclear pore complex-lamina structures in oocytes and erythrocytes of Xenopus laevis

Nuclear envelopes from oocytes of Xenopus laevis are rich in pore complexes and contain a major polypeptide of apparent molecular weight (M_r) 68,000. A rapid extraction procedure using buffer containing 1% ($\text{v}\text{v}$) Triton X-100 and 1.0 m-KCl allows the preparation of insoluble nuclear envelope skeletons showing only residual pore complex structures, with some interconnecting filament material, and one major polypeptide; i.e. that of M_r 68,000. This skeletal protein, which is not found in nuclear contents, reveals, on two-dimensional gel electrophoresis, a series of distinct isoelectric variants focusing in the pH range from 6.4 to 6.6. In living oocytes, this protein is continuously synthesized, as demonstrated by incorporation of labelled amino acids, and phosphorylated, A similar prominent skeletal protein has been found in nuclear envelopes of oocytes of other amphibia; however, slight but significant differences in electrophoretic mobility can be noted between different amphibian species.For comparison, nucleocortical lamina structures containing few pore complexes have been isolated, using similar extraction procedures, from various somatic cells of X. laevis, including erythrocytes. Laminae from these cells contain two major polypeptides, one (L_I) of M_r 72,000 focusing at approximately pH 5.35 and another (L_II) of M_r 69,000 focusing in several variants between pH 6.20 and 6.35. Similarly extracted “pore complex-lamina” fractions from rat liver contain a polypeptide of similar size and electrical charge as protein L_I from Xenopus and, in addition, two other polypeptides (M_r values: 74,000 and 62,000) both focusing between pH 6.6 and 6.9.It is concluded that the pore complex-lamina structure of the oocyte nucleus is assembled by only one major protein of M_r 68,000. The results also show that the protein composition of this insoluble nucleocortical structure can be different in different cells of the same organism. The compositional differences of these nuclear envelope skeletons are discussed in relation to the relative proportions of pore complex and interporous (lamina) material in the nuclear envelopes of the specific cells. It is suggested that the M_r 68,000 protein predominant in oocyte nuclear envelopes contributes, as an architectural component, to the formation of the highly organized nuclear pore complex.     

Architecture and polypeptide composition of HeLa cytoskeletons: Modification of cytoarchitectural polypeptides during mitosis

Substrate-attached asynchronous HeLa cells were extracted with Triton X-100 and analysed by electron microscopy and two-dimensional gel electrophoresis. Such Triton cytoskeletons showed actin filament bundles, microtubules, intermediate filaments, and actin networks in the substrate-associated lamellae, and contained around 90 polypeptides (48 basic, 42 acidic; 52% of total actin, 99% of vimentin, 41% of α-actinin and 30% of β-tubulin).Cytoskeletons produced by further extraction in high and low salt buffers (L-H-L) showed only intermediate filaments, the nucleus and residual actin, and contained a total of 19 polypeptides (13 acidic, 6 basic). Of these, 12 corresponded to abundant acidic proteins in the 47,000 to 70,000 M_r region as determined by staining with Coomassie blue and labelling with a mixture of ¹⁴C-labelled amino acids. Using L-H-L extracted cytoplasts, and employing an actin depolymerising protein from slime moulds, seven abundant acidic IEF³ polypeptides were shown to be present in these intermediate filament-enriched, substrate-attached cytoplast cytoskeletons. These polypeptides (L-H-L cytoplast polypeptides) corresponded to vimentin (IEF 26, 54,000 M_rmr) and six polypeptides (IEF 12, 68,000 M_r; IEF 24, 56,000 M_r; IEF 31, 50,000 M_r; IEF 35, 49,000 M_r; IEF 36, 48,500 M_r and IEF 46, 43,500 M_r) not previously reported as present in cytoskeletons. Peptide analysis showed that these were not related as products of modification or proteolysis.Labelling of mitotic and interphase cells with [³⁵S]methionine followed by one-dimensional peptide map analysis showed that IEF 24, 26 (vimentin), 31 and 36 are preferentially modified during mitosis. These modifications correspond to phosphorylations of IEF 26 (vimentin) and 31, and to an unknown type for IEF 24. IEF 36 is phosphorylated in interphase to yield IEF 37, and the latter is further phosphorylated in mitosis. These results suggest that modification of the L-H-L cytoplast polypeptides may be important in the reorganization of cytoskeletal elements that takes place during cell division.     

Common identity of substrate binding subunit of vacuolar h-translocating inorganic pyrophosphatase of higher plant cells

There have been conflicting reports in the literature concerning the polypeptide composition of the vacuolar H⁺-translocating inorganic pyrophosphatase (tonoplast H⁺-PPase) of plant cells. The major subunit(s) of the enzyme have been attributed to polypeptides of relative molecular weight (M_r) 64,500 (Beta vulgaris), 67,000 (Beta vulgaris), 73,000 (Vigna radiata), and 37,000 to 45,000 (Zea mays). Here, we reconcile these differences to show, through the combined application of independent purification, affinity-labeling, sequencing, and immunological procedures, that the major polypeptide associated with the H⁺-PPase from all of these organisms, and Arabidopsis thaliana, corresponds to the same moiety. The principal polypeptide components of the H⁺-PPase purified from Beta and Vigna by independent procedures have similar apparent subunit masses when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under identical conditions (M_r(Beta) = 64,500; M_r(Vigna) = 66,000) and exhibit identical kinetics of irreversible inhibition and ligand-modified labeling by [¹⁴C]-N-ethylmaleimide. Similarly, the M_r 64,500 and 67,000 polypeptides isolated from Beta by independent methods (cf. C.J. Britten, J.C. Turner, P.A. Rea [1989] FEBS Lett 256: 200-206 versus V. Sarafian and R.J. Poole [1989] Plant Physiol 91: 34-38) are indistinguishable: the two polypeptides comigrate when electrophoresed under the same conditions and yield tryptic fragments with identical overlapping sequences. Because both the N-terminal sequence of the M_r 66,000 subunit of the H⁺-PPase isolated from Vigna and the direct sequence data from Beta align precisely with the deduced amino acid sequence of cDNAs encoding the H⁺-PPase of Arabidopsis, all three enzymes are inferred to be highly conserved structurally. Accordingly, immunoblots of membranes prepared from Arabidopsis, Beta, Vigna, and Zea, probed with antibody affinity purified against the magnesium inorganic pyrophosphate-binding, M_r 66,000 polypeptide of Vigna, reveal a single immunoreactive band at M_r 64,500 to 67,000 in all four preparations. The M_r 66,000 polypeptide of Zea membranes is, however, prone to proteolysis during membrane fractionation and selective aggregation during sample denaturation for SDS-PAGE. The anomalous M_r 37,000 to 45,000 subunit pattern previously ascribed to the H⁺-PPase from Zea (A. Chanson and P.E. Pilet [1989] Plant Physiol 90: 934-938) is attributed to loss of the M_r 66,000 subunit and the appearance of polypeptide fragments of M_r 44,700 and 39,000 through the combined effects of sample aggregation before SDS-PAGE and proteolysis, respectively. It is, therefore, concluded that the substrate-binding subunit of the tonoplast H⁺-PPase has a common identity in all four organisms.     

Evidence for the presence of phospholamban in the endoplasmic reticulum of smooth muscle

In microsomal vesicles isolated from several smooth muscles many polypeptides were phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. In pig stomach and in rabbit and dog aorta components of M_r 22 000 and 11 000 were identified as forms of phospholamban. These polypeptides were, however, not observed in pig aorta. These phospholamban-like polypeptides presented the same electrophoretic mobility in sodium dodecyl sulphate gels as cardiac phospholamban, and the 22 000 M_r form showed a similar reaction to heat treatment in sodium dodecyl sulphate. Antibodies against purified canine cardiac phospholamban cross-reacted with the 22 000 and 11 000 M_r phosphorylatable polypeptides from smooth muscle membranes. Subcellular fractionation of porcine stomach smooth muslce indicated that phospholamban was present in the membranes of the endoplasmic reticulum and not in the plasma membranes. Phospholamban was also phosphorylated by an endogenous calcium—calmodulin-dependent protein kinase and by an endogenous cyclic AMP-dependent kinase. It is concluded that the endoplasmic reticulum of many, but possibly not all, smooth muscles contains phospholamban. However, the physiological role of phospholamban in smooth muscle remains to be established.     

Molecular characteristics of lipophorin,the juvenile hormone-binding protein in the hemolymph of the Colorado potato beetle

Lipophorin, the protein that specifically binds juvenile hormone in the hemolymph of the Colorado potato beetle, Leptinotarsa decemlineata, is a high-density lipoprotein of M_r ～ 574,000. Lipophorin contains 43% lipid and is composed of two apoproteins: apolipophorin I (M_r ～ 251,000) and apolipophorin II (M_r ～ 78,000). Both apoproteins contain mannose residues. Carotenoids make up a substantial part of the lipid fraction. Lipophorin constitutes about 25% of the total hemolymph proteins. Its concentration in the hemolymph (26 μM in 4-day-old long-day and 40 μM in 4-day-old short-day beetles) changes with different physiological conditions concomitant with changes in total protein content. Lipophorin specifically binds 10R-juvenile hormone III with high affinity. The dissociation constant for 10R-juvenile hormone III is 12 ± 2 nM. One lipophorin molecule contains one specific juvenile hormone-binding site. The concentration of binding sites therefore equals that of lipophorin in hemolymph.     

Biotin-mediated delivery of exogenous macromolecules into soybean cells

We have demonstrated that attachment of biotin to a variety of macromolecules allows the uptake of those macromolecules into cultured soybean cells (Glycine max Merr cv Kent). Macromolecules that were nondestructively delivered into intact cells in large numbers (>10⁶/cell) by this technique include bovine insulin (M_r about 5,700), bovine ribonuclease (M_r about 14,000), human hemoglobin (M_r about 64,000), and bovine serum albumin (M_r about 68,000). It is hypothesized that this methodology may be useful for delivering antibodies, toxins, enzymes, and genetic material into living plant cells without requiring prior removal of the cell wall or infection with Agrobacterium.     

Chloroplast thylakoid membrane proteins having buried amine buffering groups

Some chloroplast thylakoid membrane proteins have anomalously low pK_a (near 7.8) amine groups, indicating that the buffering groups may be buried in hydrophobic regions and/or close to other positive charges. Other work has shown that the low pK_a amine group array is not in ready equilibrium with either the inner or outer bulk aqueous phases (Laszlo, J.A., Baker, G.M. and Dilley, R.A. (1984) J. Bioenerg. Biomembranes, 16, 37–51). Acetic anhydride reacts with the neutral amine and has been used as a probe for labeling the low pK_a amines. The buried array of buffering groups can be labeled with [³H]acetic anhydride in the dark only after the membranes were made leaky to protons with uncoupler addition. Sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis was used to separate the polypeptides and identify those that show the uncoupler-dependent labeling increase. Included in that group are polypeptides known to be associated with Photosystem II having M_r 17000, 22000 and 31000, some of the light-harvesting pigment proteins with M_r 24000–28000, the CF₀ component with M_r 8000 and some polypeptides associated with Photosystem I. A protein with M_r 15000 showed very large changes in labeling, but the identity of this polypeptide is unknown. The arrays of buried amine buffering groups are diversely distributed among membrane proteins and it is not clear what role, if any, they play in membrane function.     

The purification and partial amino acid sequence of a polypeptide from the glutelin fraction of rice grains; homology to pea legumin

The glutelin fraction was extracted from grain meals of rice (Oryzea sativa) with 50 mM Tris-HCl buffer (pH 8.8) containing 6 M urea and 10 mM 2-mercaptoethanol. Polypeptides of glutelin were separated and purified by ion-exchange chromatography under denaturing conditions. Analysis by two-dimensional gel electrophoresis showed that 2 major polypeptides of the rice glutelin fraction, M_r 36 000 and 22 000, were linked in disulphide bonded pairs containing one M_r 36 000 and one M_r 22 000 subunit. A partial amino acid sequence of the purified M_r 22 000 glutelin subunit showed it to be homologous to the β-subunit of pea legumin, a storage protein which also contains disulphide-linked subunit pairs (M_r 38 000 and M_r 22 000). It is therefore proposed that the major component of rice glutelin is a legumin-like protein.     

Oat seed globulin: subunit characterization and demonstration of its synthesis as a precursor

The predominant storage protein of oat (Avena sativa L.) seeds is a saline-soluble globulin with a mol wt of 320,000 which is composed of six large (M_r = 35,000 to 40,000) and six small (M_r = 20,000 to 25,000) subunits. Experiments were conducted to further describe the subunit polypeptides and to identify the initial translation products of globulin mRNAs. Approximately 20 large subunits and 10 small subunits were resolved by two-dimensional gel analysis. The large and small subunits had acidic and basic isoelectric points, respectively. Disulfide-linked complexes of one large and one small subunit were isolated by extraction in buffer lacking a reducing agent. The NH₂-terminal sequence of the small subunits was homologous to a small subunit of soybean glycinin. Immunoprecipitation of in vitro translation products of poly(A)⁺ RNA with anti-oat globulin sera yielded M_r = 60,000 to 68,000 polypeptides. In vivo labeling of spikelets with radioactive amino acids resulted in high amounts of incorporation into polypeptides with M_r = 65,000 to 68,000 which were immunoprecipitated with anti-globulin sera. These two results suggest oat globulin is synthesized as a higher mol wt precursor which is subsequently processed to yield the large and small subunit polypeptides.     

Identification of antigenic components of Toxoplasma gondii by an immunoblotting technique

Proteins of Toxoplasma gondii were separated by SDS-polyacrylamide gel electrophoresis with subsequent transfer to a nitrocellulose sheet by electrophoretic blotting. Immunologically reactive polypeptides were detected by human sera with previously known toxoplasma antibody levels. Heavy chain-specific, peroxidase-conjugated anti-human immunoglobulins were used as the indicator antibodies for the separate identification of IgG and IgM reactive polypeptides. IgG toxoplasma antibodies reacted with several antigens of M_r ≈27 000–67 000, while toxoplasma-specific IgM seemed to detect only a few polypeptides. The M_r of 35 000 for the dominating IgM reactive polypeptide was observed.     

Purification and molecular properties of 3 polypeptides released from a highly active O2-evolving photosystem-II preparation by Tris-treatment

Tris-treatment of a highly active O₂-evolving photosystem-II preparation induced release of 3 polypeptides (M_r 33 000, 24 000 and 18 000), concomitant with inhibition of O₂ evolution [FEBS Lett. (1981)_133. 265-268]. The 3 polypeptides were purified with the use of electrofocusing. Isoelectric points of the proteins were 5.1, 6.5 and 9.2 in order of decreasing M_r value. Only a trace amount of histidine, cystein and methionine were detected in these proteins. Based on the amino acid compositions, polarity indexes of the proteins were calculated to be 47–49%, suggesting the 3 proteins to be hydrophilic.