The invariant system of coordinates of antibody molecules: prediction of the "standard" C alpha framework of VL and VH domains.

A new approach of comparing protein structures that does not involve the procedure of superposition is suggested. An invariant system of coordinates for immunoglobulin molecules that is based on the geometrical symmetry inherent to the variable domain light-chain (VL)-heavy-chain (VH) complex is described. The coordinates of the Calpha atoms in 22 immunoglobulin structures are calculated in the invariant system of coordinates. We found that 76 identical positions in this Calpha framework are symmetrical about the twofold axis. Comparison of the identical positions in these molecules allows us to select 96 positions in the light chains and 87 positions in the heavy chains whose Calpha atom coordinates are approximately the same. To check whether the average coordinates of Calpha atoms in these positions complies with the stereochemical requirements, we calculated Calpha-Calpha distances. Seventy-three positions of the light chains and 72 positions of the heavy chains satisfy the Calpha-Calpha distance criterion. The Calpha atoms in these positions are used for constructing the "standard" Calpha framework of VL and VH complexes. The average coordinates of Calpha atoms are presented.

A shark antibody heavy chain encoded by a nonsomatically rearranged VDJ is preferentially expressed in early development and is convergent with mammalian IgG

In most vertebrate embryos and neonates studied to date unique antigen receptors (antibodies and T cell receptors) are expressed that possess a limited immune repertoire. We have isolated a subclass of IgM, IgM1gj, from the nurse shark Ginglymostoma cirratum that is preferentially expressed in neonates. The variable (V) region gene encoding the heavy (H) chain underwent V-D-J rearrangement in germ cells (“germline-joined”). Such H chain V genes were discovered over 10 years ago in sharks but until now were not shown to be expressed at appreciable levels; we find expression of H1gj in primary and secondary lymphoid tissues early in life, but in adults only in primary lymphoid tissue, which is identified in this work as the epigonal organ. H1gj chain associates covalently with light (L) chains and is most similar in sequence to IgM H chains, but like mammalian IgG has three rather than the four IgM constant domains; deletion of the ancestral IgM C2 domain thus defines both IgG and IgM1gj. Because sharks are the members of the oldest vertebrate class known to possess antibodies, unique or specialized antibodies expressed early in ontogeny in sharks and other vertebrates were likely present at the inception of the adaptive immune system.

Random circular permutation of genes and expressed polypeptide chains: application of the method to the catalytic chains of aspartate transcarbamoylase.

Recent studies on proteins whose N and C termini are in close proximity have demonstrated that folding of polypeptide chains and assembly of oligomers can be accomplished with circularly permuted chains. As yet no methodical study has been conducted to determine how extensively new termini can be introduced and where such termini cannot be tolerated. We have devised a procedure to generate random circular permutations of the catalytic chains of Escherichia coli aspartate transcarbamoylase (ATCase; EC 2.1.3.2) and to select clones that produce active or stable holoenzyme containing permuted chains. A tandem gene construct was made, based on the desired linkage between amino acid residues in the C- and N-terminal regions of the polypeptide chain, and this DNA was treated with a suitable restriction enzyme to yield a fragment containing the rearranged coding sequence for the chain. Circularization achieved with DNA ligase, followed by linearization at random with DNase I, and incorporation of the linearized, repaired, blunt-ended, rearranged genes into a suitable plasmid permitted the expression of randomly permuted polypeptide chains. The plasmid with appropriate stop codons also contained pyrI, the gene encoding the regulatory chain of ATCase. Colonies expressing detectable amounts of ATCase-like molecules containing permuted catalytic chains were identified by an immunoblot technique or by their ability to grow in the absence of pyrimidines in the growth medium. Sequencing of positive clones revealed a variety of novel circular permutations. Some had N and C termini within helices of the wild-type enzyme as well as deletions and insertions. Permutations were concentrated in the C-terminal domain and only few were detected in the N-terminal domain. The technique, which is adaptable generally to proteins whose N and C termini are near each other, can be of value in relating in vivo folding of nascent, growing polypeptide chains to in vitro renaturation of complete chains and determining the role of protein sequence in folding kinetics.

Direct measurement of salt-bridge solvation energies using a peptide model system: implications for protein stability.

The solvation energies of salt bridges formed between the terminal carboxyl of the host pentapeptide AcWL- X-LL and the side chains of Arg or Lys in the guest (X) position have been measured. The energies were derived from octanol-to-buffer transfer free energies determined between pH 1 and pH 9. 13C NMR measurements show that the salt bridges form in the octanol phase, but not in the buffer phase, when the side chains and the terminal carboxyl group are charged. The free energy of salt-bridge formation in octanol is approximately -4 kcal/mol (1 cal = 4.184 J), which is equal to or slightly larger than the sum of the solvation energies of noninteracting pairs of charged side chains. This is about one-half the free energy that would result from replacing a charge pair in octanol with a pair of hydrophobic residues of moderate size. Therefore, salt bridging in octanol can change the favorable aqueous solvation energy of a pair of oppositely charged residues to neutral or slightly unfavorable but cannot provide the same free energy decrease as hydrophobic residues. This is consistent with recent computational and experimental studies of protein stability.

Roles of heavy and light chains in IgM polymerization.

IgM antibodies are secreted as multisubunit polymers that consist of as many as three discrete polypeptides: mu heavy chains, light (L) chains, and joining (J) chains. We wished to determine whether L chains that are required to confer secretory competence on immunoglobulin molecules must be present for IgM to polymerize--that is, for intersubunit disulfide bonds to form between mu chains. Using a L-chain-loss variant of an IgM-secreting hybridoma, we demonstrated that mu chains were efficiently polymerized independent of L chains, in a manner similar to that observed for conventional microL complexes, and that the mu polymers incorporated J chain. These mu polymers were not secreted but remained associated with the endoplasmic reticulum-resident chaperone BiP (GRP78). This finding is consistent with the endoplasmic reticulum being the subcellular site of IgM polymerization. We conclude that mu chain alone has the potential to direct the polymerization of secreted IgM, a process necessary but not sufficient for IgM to attain secretory competence.