Synthesis and export of the outer membrane lipoprotein in Escherichia coli mutants defective in generalized protein export.

Export of the outer membrane lipoprotein in Escherichia coli was examined in conditionally lethal mutants that were defective in protein export in general, including secA, secB, secC, and secD. Lipoprotein export was affected in a secA(Ts) mutant of E. coli at the nonpermissive temperature; it was also affected in a secA(Am) mutant of E. coli at the permissive temperature, but not at the nonpermissive temperature. The export of lipoprotein occurred normally in E. coli carrying a null secB::Tn5 mutation; on the other hand, the export of an OmpF::Lpp hybrid protein, consisting of the signal sequence plus 11 amino acid residues of mature OmpF and mature lipoprotein, was affected by the secB mutation. The synthesis of lipoprotein was reduced in the secC mutant at the nonpermissive temperature, as was the case for synthesis of the maltose-binding protein, while the synthesis of OmpA was not affected. Lipoprotein export was found to be slightly affected in secD(Cs) mutants at the nonpermissive temperature. These results taken together indicate that the export of lipoprotein shares the common requirements for functional SecA and SecD proteins with other exported proteins, but does not require a functional SecB protein. SecC protein (ribosomal protein S15) is required for the optimal synthesis of lipoprotein.     

Regions of maltose-binding protein that influence SecB-dependent and SecA-dependent export in Escherichia coli.

In Escherichia coli, the efficient export of maltose-binding protein (MBP) is dependent on the chaperone SecB, whereas export of ribose-binding protein (RBP) is SecB independent. To localize the regions of MBP involved in interaction with SecB, hybrids between MBP and RBP in SecB mutant cells were constructed and analyzed. One hybrid consisted of the signal peptide and first third of the mature moiety of MBP, followed by the C-terminal two-thirds of RBP (MBP-RBP112). This hybrid was dependent upon SecB for its efficient export and exhibited a strong export defect in secA mutant cells. A hybrid between RBP and MBP with the same fusion point was also constructed (RBP-MBP116). The RBP-MBP116 hybrid remained SecB independent and only exhibited a partial export defect in secA mutant cells. In addition, MBP species with specific alterations in the early mature region were less dependent on SecB for their efficient export. The export of these altered MBP species was also less affected in secA mutant cells and in cells treated with sodium azide. These results present additional evidence for the targeting role of SecB.     

SecB-independent export of Escherichia coli ribose-binding protein (RBP): some comparisons with export of maltose-binding protein (MBP) and studies with RBP-MBP hybrid proteins.

The efficient export of the Escherichia coli maltose-binding protein (MBP) is known to be SecB dependent, whereas ribose-binding protein (RBP) export is SecB independent. When the MBP and RBP signal peptides were exchanged precisely at the signal peptidase processing sites, the resultant RBP-MBP and MBP-RBP hybrid proteins both were efficiently exported in SecB+ cells. However, only MBP-RBP was efficiently exported in SecB- cells; RBP-MBP exhibited a significant export defect, a finding that was consistent with previous proposals that SecB specifically interacts with the mature moiety of precursor MBP to promote export. The relatively slow, totally posttranslational export mode exhibited by certain mutant RBP and MBP-RBP species in SecB+ cells was not affected by the loss of SecB. In contrast, MBP and RBP-MBP species with similarly altered signal peptides were totally export defective in SecB- cells. Both export-defective MBP and RBP-MBP interfered with SecB-mediated protein export by depleting cells of functional SecB. In contrast, neither export-defective RBP nor MBP-RBP elicited such an interference effect. These and other data indicated that SecB is unable to interact with precursor RBP or that any interaction between these two proteins is considerably weaker than that of SecB with precursor MBP. In addition, no correlation could be established between a SecB requirement for export and PrlA-mediated suppression of signal peptide export defects. Finally, previous studies have established that wild-type MBP export can be accomplished cotranslationally, whereas wild-type RBP export is strictly a posttranslational process. In this study, cotranslational export was not detected for either MBP-RBP or RBP-MBP. This indicates that the export mode exhibited by a given precursor protein (cotranslational versus posttranslational) is determined by properties of both the signal peptide and the mature moiety.     

The antifolding activity of SecB promotes the export of the E. coli maltose-binding protein

Evidence is presented that the E. coli secB gene encodes a soluble protein that interacts with the mature region of the precursor maltose-binding protein (MBP), and promotes MBP export by preventing premature folding of the newly synthesized polypeptide into an export-incompetent form. The interaction of SecB with MBP was indicated by the finding that synthesis of various export-defective MBP species interfered with normal protein export by limiting SecB availability. The antifolding activity of SecB was demonstrated by the following: the defect in MBP export in SecB- cells was suppressed by mutational alterations affecting MBP folding; export of a mutant MBP that is accomplished in a strictly posttranslational mode was totally blocked in SecB- cells; and the rate of folding of wild-type MBP synthesized in vitro was found to be accelerated when SecB was absent and greatly retarded when excess SecB was present.     

Effects of Escherichia coli secB mutations on pre-maltose binding protein conformation and export kinetics

Mutations affecting the secB gene of Escherichia coli cause a defect in protein export. This report presents the demonstration that the secB mutations caused a defect in co-translational processing of maltose binding protein (MBP). A significant amount of post-translational processing of pre-MBP occurred within 1 min after termination of pulse labeling; at later time points only a small amount of additional processing occurred. The conformation of the intracellular precursor form of MBP was examined in a secB::Tn5 mutant, using protease sensitivity (Randall, L. L., and Hardy, S. J. S. (1986) Cell 46, 921-928) as the assay. In contrast to the isogenic wild type strain, a population of pre-MBP that had folded into a protease resistant conformation was detected in the secB mutant. In addition, sublethal doses of chloramphenicol did not significantly affect protein export in the secB::Tn5 mutant and the secB::Tn5 mutation did not lead to defects in membrane energization.     

Alterations in the hydrophilic segment of the maltose-binding protein (MBP) signal peptide that affect either export or translation of MBP.

Mutations that reduce the net positive charge within the hydrophilic segments of the signal peptides of several prokaryotic exported proteins can result in a reduction in the rate of protein export, as well as a reduction in protein synthesis (M. N. Hall, J. Gabay, and M. Shwartz, EMBO J. 2:15-19, 1983; S. Inouye, X. Soberon, T. Franceschini, K. Nakamura, K. Itakura, and M. Inouye, Proc. Natl. Acad. Sci. USA 79:3438-3441, 1982; J. W. Puziss, J. D. Fikes, and P. J. Bassford, Jr., J. Bacteriol. 171:2302-2311, 1989). This result has been interpreted as evidence that the hydrophilic segment is part of a mechanism that obligatorily couples translation to protein export. We have investigated the role of the hydrophilic segment of the Escherichia coli maltose-binding protein (MBP) signal peptide in the export and synthesis of MBP. Deletion of the entire hydrophilic segment from the MBP signal peptide resulted in a defect in MBP export, as well as a dramatic reduction in total MBP synthesis. Suppressor mutations that lie upstream of the malE coding region were isolated. These mutations do not affect MBP export but instead were shown to partially restore MBP synthesis by increasing the efficiency of MBP translational initiation. In addition, analysis of a series of substitution mutations in the second codon of certain malE alleles demonstrated that MBP export and synthesis can be independently affected by mutations in the hydrophilic segment. Finally, analysis of alterations in the hydrophilic segment of the ribose-binding protein signal peptide fused to the mature moiety of the MBP has revealed that the role of the hydrophilic segment in the export process can be functionally separated from any role in translation. Taken together, these results strongly suggest that the hydrophilic segment of the MBP signal peptide is not involved in a mechanism that couples MBP translation to export and argue against the presence of a mechanism that obligatorily couples translation to protein export in Escherichia coli.     

Export and processing of MalE-LacZ hybrid proteins in Escherichia coli

Five classes of MalE-LacZ hybrid proteins have previously been characterized. These proteins differ in the amount of the maltose-binding protein (MBP) that is attached to beta-galactosidase. Although none of these proteins is secreted into the periplasm, the four larger classes of hybrid proteins, those that include an intact MBP signal peptide, are inserted into the cytoplasmic membrane, suggesting that the secretion process has at least been initiated. In this study, we demonstrated that some portion of the four larger hybrid proteins can be translocated across the cytoplasmic membrane, thus permitting processing of the signal peptide. We have found that hybrid proteins that include only a small portion of the mature MBP are inefficiently recognized as exported proteins, and translocation and processing of these appear to be relatively slow, posttranslational events. In marked contrast, hybrid proteins that include a substantial portion of the mature MBP are efficiently recognized, and translocation and processing of these occur very rapidly, possibly cotranslationally. Our results complement other studies and very strongly suggest a role for the mature MBP in the export process.     

Export pathway selectivity of Escherichia coli twin arginine translocation signal peptides

The Escherichia coli genome encodes at least 29 putative signal peptides containing a twin arginine motif characteristic of proteins exported via the twin arginine translocation (Tat) pathway. Fusions of the putative Tat signal peptides plus six to eight amino acids of the mature proteins to three reporter proteins (short-lived green fluorescent protein, maltose-binding protein (MBP), and alkaline phosphatase) and also data from the cell localization of epitope-tagged full-length proteins were employed to determine the ability of the 29 signal peptides to direct export through the Tat pathway, through the general secretory pathway (Sec), or through both. 27/29 putative signal peptides could export one or more reporter proteins through Tat. Of these, 11 signal peptides displayed Tat specificity in that they could not direct the export of Sec-only reporter proteins. The rest (16/27) were promiscuous and were capable of directing export of the appropriate reporter either via Tat (green fluorescent protein, MBP) or via Sec (PhoA, MBP). Mutations that conferred a >or=+1 charge to the N terminus of the mature protein abolished or drastically reduced routing through the Sec pathway without affecting the ability to export via the Tat pathway. These experiments demonstrate that the charge of the mature protein N terminus affects export promiscuity, independent of the effect of the folding state of the mature protein.     

Evidence for specificity at an early step in protein export in Escherichia coli.

We previously described mutations in a gene, secB, which have pleiotropic effects on protein export in Escherichia coli. In this paper, we report the isolation of mutants in which the activity of the secB gene was eliminated. Null mutations in secB affected only a subset of exported proteins. Strains carrying these mutations, although unable to grow on L broth plates, were still viable on minimal media. These secB mutations reversed a block in the translation of an exported protein that was caused by the elimination of another component of the secretion machinery, SecA protein. These results suggest that the secB product acts at an early step in the export process and is involved in the export of only a subset of cell envelope proteins.     

Nuclear retention of MBP mRNAs in the quaking viable mice

Quaking viable (qk(v)) mice fail to properly compact myelin in their central nervous systems. Although the defect in the qk(v) mice involves a mutation affecting the expression of the alternatively spliced qk gene products, their roles in myelination are unknown. We show that the QKI RNA binding proteins regulate the nuclear export of MBP mRNAs. Disruption of the QKI nucleocytoplasmic equilibrium in oligodendrocytes results in nuclear and perikaryal retention of the MBP mRNAs and lack of export to cytoplasmic processes, as it occurs in qk(v) mice. MBP mRNA export defect leads to a reduction in the MBP levels and their improper cellular targeting to the periphery. Our findings suggest that QKI participates in myelination by regulating the mRNA export of key protein components.     

Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm.

We have employed the technique of gene fusion to fuse the LacZ gene encoding the cytoplasmic enzyme beta-galactosidase with the malE gene encoding the periplasmic maltose binding protein (MBP). Strains were obtained which synthesize malE-lacZ hybrid proteins of various sizes. These proteins have, at their amino terminus, a portion of the MBP and at their carboxyl terminus, enzymatically active beta-galactosidase. When the hybrid protein includes only a small, amino-terminal portion of the MBP, the hybrid protein residues in the cytoplasm. When the hybrid protein contains enough of the MBP to include an intact MBP signal sequence, a significant portion of the hybrid protein is found in the cytoplasmic membrane, suggesting that secretion of the hybrid protein has been initiated. However, in no case is the hybrid protein secreted into the periplasm, even when the hybrid protein includes almost the entire MBP. In the latter case, the synthesis and attempted export of the hybrid protein interferes with the export of at least certain normal envelope proteins, which accumulate in the cell in their precursor forms, and the cell dies. These results suggest that a number of envelope proteins may be exported at a common site, and that there are only a limited number of such sites. Also, these results indicate that it is not sufficient to simply attach an amino-terminal signal sequence to a polypeptide to assure its export.     

Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations.

It is believed that one or more basic residues at the extreme amino terminus of precursor proteins and the lack of a net positive charge immediately following the signal peptide act as topological determinants that promote the insertion of the signal peptide hydrophobic core into the cytoplasmic membrane of Escherichia coli cells with the correct orientation required to initiate the protein export process. The export efficiency of precursor maltose-binding protein (pre-MBP) was found to decrease progressively as the net charge in the early mature region was increased systematically from 0 to +4. This inhibitory effect could be further exacerbated by reducing the net charge in the signal peptide to below 0. One such MBP species, designated MBP-3/+3 and having a net charge of -3 in the signal peptide and +3 in the early mature region, was totally export defective. Revertants in which MBP-3/+3 export was restored were found to harbor mutations in the prlA (secY) gene, encoding a key component of the E. coli protein export machinery. One such mutation, prlA666, was extensively characterized and shown to be a particularly strong suppressor of a variety of MBP export defects. Export of MBP-3/+3 and other MBP species with charge alterations in the early mature region also was substantially improved in E. coli cells harboring certain other prlA mutations originally selected as extragenic suppressors of signal sequence mutations altering the hydrophobic core of the LamB or MBP signal peptide. In addition, the enzymatic activity of alkaline phosphatase (PhoA) fused to a predicted cytoplasmic domain of an integral membrane protein (UhpT) increased significantly in cells harboring prlA666. These results suggest a role for PrlA/SecY in determining the orientation of signal peptides and possibly other membrane-spanning protein domains in the cytoplasmic membrane.     

Thirty-three amino acids of the mature moiety of an unprocessed maltose-binding protein are sufficient for export in Escherichia coli.

Maltose-binding protein (MBP) is translocated across the cytoplasmic membrane of Escherichia coli; successful export depends on information in both the signal peptide and the mature moiety of the protein. To determine the shortest portion of the mature region that would maintain detectable entry of MBP into the export pathway, we took advantage of the properties of an MBP species with proline substituted in the +1 position relative to the cleavage site (MBP27-P). This protein efficiently crosses the cytoplasmic membrane but is not processed and acts as a competitive inhibitor of signal peptidase I (leader peptidase). Export of MBP27-P is measured by the inhibition of processing of other proteins, such as ribose-binding protein (RBP). A series of truncated derivatives of MBP27-P were tested for the ability to inhibit processing of RBP. An MBP27-P species with only 33 amino acids of the mature moiety inhibited processing of RBP, indicating that this truncated polypeptide was probably exported and interacted with signal peptidase I.     

Regulation of cell proliferation by nucleocytoplasmic dynamics of postnatal and embryonic exon-II-containing MBP isoforms

The only known structural protein required for formation of myelin, produced by oligodendrocytes in the central nervous system, is myelin basic protein (MBP). This peripheral membrane protein has different developmentally-regulated isoforms, generated by alternative splicing. The isoforms are targeted to distinct subcellular locations, which is governed by the presence or absence of exon-II, although their functional expression is often less clear. Here, we investigated the role of exon-II-containing MBP isoforms and their link with cell proliferation. Live-cell imaging and FRAP analysis revealed a dynamic nucleocytoplasmic translocation of the exon-II-containing postnatal 21.5-kDa MBP isoform upon mitogenic modulation. Its nuclear export was blocked upon treatment with leptomycin B, an inhibitor of nuclear protein export. Next to the postnatal MBP isoforms, embryonic exon-II-containing MBP (e-MBP) is expressed in primary (immature) oligodendrocytes. The e-MBP isoform is exclusively present in OLN-93 cells, a rat-derived oligodendrocyte progenitor cell line, and interestingly, also in several non-CNS cell lines. As seen for postnatal MBPs, a similar nucleocytoplasmic translocation upon mitogenic modulation was observed for e-MBP. Thus, upon serum deprivation, e-MBP was excluded from the nucleus, whereas re-addition of serum re-established its nuclear localization, with a concomitant increase in proliferation. Knockdown of MBP by shRNA confirmed a role for e-MBP in OLN-93 proliferation, whereas the absence of e-MBP similarly reduced the proliferative capacity of non-CNS cell lines. Thus, exon-II-containing MBP isoforms may regulate cell proliferation via a mechanism that relies on their dynamic nuclear import and export, which is not restricted to the oligodendrocyte lineage.     

Export of the periplasmic maltose-binding protein ofEscherichia coli

The export of the maltose-binding protein (MBP), themalE gene product, to the periplasm ofEschericha coli cells has been extensively investigated. The isolation of strains synthesizing MalE-LacZ hybrid proteins led to a novel genetic selection for mutants that accumulate export-defective precursor MBP (preMBP) in the cytoplasm. The export defects were subsequently shown to result from alterations in the MBP signal peptide. Analysis of these and a variety of mutants obtained in other ways has provided considerable insight into the requirements for an optimally functional MBP signal peptide. This structure has been shown to have multiple roles in the export process, including promoting entry of preMBP into the export pathway and initiating MBP translocation across the cytoplasmic membrane. The latter has been shown to be a late event relative to synthesis and can occur entirely posttranslationally, even many minutes after the completion of synthesis. Translocation requires that the MBP polypeptide exist in an export-competent conformation that most likely represents an unfolded state that is not inhibitory to membrane transit. The signal peptide contributes to the export competence of preMBP by slowing the rate at which the attached mature moiety folds. In addition, preMBP folding is thought to be further retarded by the binding of a cytoplasmic protein, SecB, to the mature moiety of nascent preMBP. In cells lacking this antifolding factor, MBP export represents a race between delivery of newly synthesized, export-competent preMBP to the translocation machinery in the cytoplasmic membrane and folding of preMBP into an export-incompetent conformation. SecB is one of threeE. coli proteins classified as molecular chaperones by their ability to stabilize precursor proteins for membrane translocation.     

Mutations that improve export of maltose-binding protein in SecB- cells of Escherichia coli.

It previously has been proposed that the Escherichia coli SecB protein promotes the export of the maltose-binding protein (MBP) from the cytoplasm by preventing the folding of the precursor MBP (preMBP) into a translocation-incompetent conformation. The export of wild-type MBP is only partially blocked in SecB- cells. In contrast, the export of MBP16-1, an MBP species with a defective signal peptide, is totally dependent on SecB; hence, SecB- cells that synthesize MBP16-1 are unable to utilize maltose as a sole carbon source. The selection of Mal+ revertants primarily yielded mutants with alterations in the MBP16-1 signal peptide that permitted SecB-independent MBP export to the periplasm to various extents. Although each of these alterations increased the overall hydrophobicity of the signal peptide, it was not possible to strictly equate changes in hydrophobicity with the degree of SecB-independent export. Somewhat unexpectedly, two mutants were obtained in which MBP export in SecB- cells was markedly superior to that of the wild-type MBP. Although wild-type MBP is not cotranslationally translocated in SecB- cells, the two mutant proteins designated MBP172 and MBP173 exhibited significant cotranslational export in the absence of SecB. Thus, the role of SecB was partially supplanted by a signal peptide that promoted more rapid movement of MBP through the export pathway. When preMBP included the MBP172 signal peptide as well as an alteration in the mature moiety that slows folding, the SecB requirement for maximal MBP export efficiency was almost totally eliminated. These results provide additional strong support for the proposed antifolding role of SecB in MBP export.     

Export of Escherichia coli alkaline phosphatase attached to an integral membrane protein, SecY

The SecY protein is a membrane-bound factor required for bacterial protein export and embedded in the cytoplasmic membrane by its 10 transmembrane segments. We previously proposed a topology model for this protein by adapting the Manoil-Beckwith TnphoA approach, a genetic method to assign local disposition of a membrane protein from the enzymatic activity of the alkaline phosphatase (PhoA) mature sequence attached to the various regions. SecY-PhoA hybrid proteins with the PhoA domain exported to the periplasmic side of the membrane have been obtained at the five putative periplasmic domains of the SecY sequence. We now extended this method to apply it to follow export of the newly synthesized PhoA domain. Trypsin treatment of detergent-solubilized cell extracts digested the internalized (unfolded) PhoA domain but not those exported and correctly folded. One of the hybrid proteins was cleaved in vivo after export to the periplasm, providing a convenient indication for the export. Results of these analyses indicate that export of the PhoA domain attached to different periplasmic regions of SecY occurs rapidly and requires the normal functioning of the secY gene supplied in trans. Thus, this membrane protein with multiple transmembrane segments contains multiple export signals which can promote rapid and secY-dependent export of the PhoA mature sequence attached to the carboxyl-terminal sides.     

Green fluorescent protein functions as a reporter for protein localization in Escherichia coli

The use of green fluorescent protein (GFP) as a reporter for protein localization in Escherichia coli was explored by creating gene fusions between malE (encoding maltose-binding protein [MBP]) and a variant of gfp optimized for fluorescence in bacteria (GFPuv). These constructs encode hybrid proteins composed of GFP fused to the carboxy-terminal end of MBP. Fluorescence was not detected when the hybrid protein was synthesized with the MBP signal sequence. In contrast, when the MBP signal sequence was deleted, fluorescence was observed. Cell fractionation studies showed that the fluorescent MBP-GFP hybrid protein was localized in the cytoplasm, whereas the nonfluorescent version was localized to the periplasmic space. Smaller MBP-GFP hybrid proteins, however, exhibited abnormal fractionation. Expression of the gene fusions in different sec mutants, as well as signal sequence processing assays, confirmed that the periplasmically localized hybrid proteins were exported by the sec-dependent pathway. The distinction between fluorescent and nonfluorescent colonies was exploited as a scorable phenotype to isolate malE signal sequence mutations. While expression of hybrid proteins comprised of full-length MBP did not result in overproduction lethality characteristic of some exported beta-galactosidase hybrid proteins, synthesis of shorter, exported hybrid proteins was toxic to the cells. Purification of MBP-GFP hybrid protein from the different cellular compartments indicated that GFP is improperly folded when localized outside of the cytoplasm. These results suggest that GFP could serve as a useful reporter for genetic analysis of bacterial protein export and of protein folding.     

Antipeptide antibodies reveal interrelationships of MBP 200 and MBP 235: unique apoB-specific receptors for triglyceride-rich lipoproteins on human monocyte-macrophages

Two human monocyte-macrophage (HMM) membrane binding proteins, (MBP) 200 and 235, are receptor candidates that bind to the apolipoprotein (apo)B-48 domain in triglyceride-rich lipoproteins for uptake independent of apoE. Microsequence analysis of the purified reduced MBP 200R characterized tryptic peptides of MBP 200R. A synthetic peptide mimicking a unique, unambiguous 10-residue sequence (AEGLMVTGGR) induced antipeptide antibodies that specifically recognized MBP 200, 235 and 200R, in 1- and 2-dimensional analyses, indicating 1) the ligand binding protein was sequenced and 2) MBP 200 and 235 yielded MBP 200R upon reduction. These antibodies identified the MBPs in human blood-borne, THP-1, U937 MMs, and endothelial cells (EC) but not in human fibroblasts or Chinese hamster ovary (CHO) cells. Fluorescence activated cell sorting (FACS) analysis located the MBPs on the MM surface as necessary for receptor function. The 10-residue, unambiguous MBP 200-derived sequence is unique, with no matches in extant protein databases. Antipeptide antibodies bind to the MBPs in reticuloendothelial cells that have this receptor activity, but not to proteins in cells that lack this receptor activity. These studies provide the first direct protein sequence and immunochemical data that a new, unique apoB receptor for triglyceride-rich lipoproteins exists in human monocytes, macrophages, and endothelial cells.     

How human IgGs against myelin basic protein (MBP) recognize oligopeptides and MBP

Myelin basic protein (MBP) is a major protein of myelin‐proteolipid shell of axons, and it plays an important role in pathogenesis of multiple sclerosis. In the literature, there are no data on how antibodies recognize different protein antigens including MBP. A stepwise increase in ligand complexity was used to estimate the relative contributions of virtually every amino acid residue (AA) of a specific 12‐mer LSRFSWGAEGQK oligopeptide corresponding to immunodominant sequence of MBP to the light chains and to intact anti‐MBP IgGs from sera of patients with multiple sclerosis. It was shown that the minimal ligands of the light chains of IgGs are many different free AAs (K _d = 0.51–0.016 M), and each free AA interacts with the specific subsite of the light chain intended for recognition of this AA in specific LSRFSW oligopeptide. A gradual transition from Leu to LSRFSWGAEGQK leads to an increase in the affinity from 10⁻¹ to 2.3 × 10⁻⁴ M because of additive interactions of the light chain with 6 AAs of this oligopeptide and then the affinity reaches plateau. The contributions of 6 various AAs to the affinity of the oligopeptide are different (K _d, M): 0.71 (S), 0.44 (R), 0.14 (F), 0.17 (S), and 0.62 (W). Affinity of nonspecific oligopeptides to the light chains of IgGs is significantly lower. Intact MBP interacts with both light and heavy chains of IgGs demonstrating 192‐fold higher affinity than the specific oligopeptide. It is a first quantitative analysis of the mechanism of proteins recognition by antibodies. The thermodynamic model was constructed to describe the interactions of IgGs with MBP. The data obtained can be very useful for understanding how antibodies against many different proteins can recognize these proteins.