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Polyamines are known to mediate diverse biological processes, and specifically to bind and stabilize compact conformations of nucleic acids, acting as chemical chaperones that promote folding by offsetting the repulsive negative charges of the phosphodiester backbone. However, whether and how polyamines modulate the structure and function of proteins remain unclear. In particular, early proteins are thought to have been highly acidic, like nucleic acids, due to a scarcity of basic amino acids in the prebiotic context. Perhaps polyamines, the abiotic synthesis of which is simple, could have served as chemical chaperones for such primordial proteins? We replaced all lysines of an ancestral 60-residue helix-bundle protein with glutamate, resulting in a disordered protein with 21 glutamates in total. Polyamines efficiently induce folding of this hyperacidic protein at submillimolar concentrations, and their potency scaled with the number of amine groups. Compared to cations, polyamines were several orders of magnitude more potent than Na+, while Mg2+ and Ca2+ had an effect similar to that of a diamine, inducing folding at approximately seawater concentrations. We propose that (i) polyamines and dications may have had a role in promoting folding of early proteins devoid of basic residues and (ii) coil–helix transitions could be the basis of polyamine regulation in contemporary proteins.

NMR detection of intermolecular interactions between protons in large protein complexes is very challenging because it is difficult to distinguish between weak NOEs from intermolecular interactions and the much larger number of strong intramolecular NOEs. This challenging task is exacerbated by the decrease in signal-to-noise ratio in the often used isotope-edited and isotope-filtered experiments as a result of enhanced T(2) relaxation. Here, we calculate a double difference spectrum that shows exclusively intermolecular NOEs and manifests the good signal-to-noise ratio in 2D homonuclear NOESY spectra even for large proteins. The method is straightforward and results in a complete picture of all intermolecular interactions involving non exchangeable protons. Ninety-seven such (1)H-(1)H NOEs were assigned for the 44 KDa interferon-α2/IFNAR2 complex and used for docking these two proteins. The symmetry of the difference spectrum, its superb resolution, and unprecedented signal-to-noise ratio in this large protein/receptor complex suggest that this method is generally applicable to study large biopolymeric complexes.

The third variable region (V3) of the HIV-1 envelope glycoprotein gp120 is a target for virus neutralizing antibodies. The V3 sequence determines whether the virus will manifest R5 or X4 phenotypes and use the CCR5 or CXCR4 chemokine coreceptor, respectively. Previous NMR studies revealed that both R5- and X4-V3 peptides bound to antibodies 0.5beta and 447-52D form beta-hairpin conformations with the GPGR segment at the turn. In contrast, in their free form, linear V3 peptides and a cyclic peptide consisting of the entire 35-residue V3 loop were highly unstructured in aqueous solution. Herein we evaluated a series of synthetic disulfide constrained V3-peptides in which the position of the disulfide bonds, and therefore the ring size, was systematically varied. NMR structures determined for singly and doubly disulfide constrained V3-peptides in aqueous solution were compared with those found for unconstrained V3(JRFL) and V3(IIIB) peptides bound to 447-52D and to 0.5beta, respectively. Our study indicated that cyclic V3 peptides manifested significantly reduced conformational space compared to their linear homologues and that in all cases cyclic peptides exhibited cross-strand interactions suggestive of beta-hairpin-like structures. Nevertheless, the singly constrained V3-peptides retained significant flexibility and did not form an idealized beta-hairpin. Incorporation of a second disulfide bond results in significant overall rigidity, and in one case, a structure close to that of V3(MN) peptide bound to 447-52D Fab was assumed and in another case a structure close to that formed by the linear V3(IIIB) peptide bound to antibody 0.5beta was assumed.

Two-dimensional nuclear magnetic resonance (2D NMR) provides one of the foremost contemporary tools available for the elucidation of molecular structure, function, and dynamics. Execution of a 2D NMR experiment generally involves scanning a series of time-domain signals S(t(2)), as a function of a t(1) time variable which undergoes parametric incrementation throughout independent experiments. Very recently, we proposed and demonstrated a general approach whereby this serial mode of data acquisition is parallelized, enabling the acquisition of complete bidimensional NMR data sets via the recording of a single transient. The present paper discusses in more detail various conceptual and experimental aspects of this novel 2D NMR methodology. The basic principles of the approach are reviewed, various homo- and heteronuclear NMR applications are illustrated, and the main features and artifacts affecting the method are derived. Extensions to higher-dimensional experiments are also briefly noted.

The alpha-subunit of the nicotinic acetylcholine receptor (alphaAChR) contains a binding site for alpha-bungarotoxin (alpha-BTX), a snake-venom-derived alpha-neurotoxin. Previous studies have established that the segment comprising residues 173-204 of alphaAChR contains the major determinant interacting with the toxin, but the precise boundaries of this determinant have not been clearly defined to date. In this study, we applied NMR dynamic filtering to determine the exact sequence constituting the major alphaAChR determinant interacting with alpha-BTX. Two overlapping synthetic peptides corresponding to segments 179-200 and 182-202 of the alphaAChR were complexed with alpha-BTX. HOHAHA and ROESY spectra of these complexes acquired with long mixing times highlight the residues of the peptide that do not interact with the toxin and retain considerable mobility upon binding to alpha-BTX. These results, together with changes in the chemical shifts of the peptide protons upon complex formation, suggest that residues 184-200 form the contact region. At pH 4, the molecular mass of the complex determined by dynamic light scattering (DLS) was found to be 11.2 kDa, in excellent agreement with the expected molecular mass of a 1:1 complex, while at pH5 the DLS measurement of 20 kDa molecular mass indicated dimerization of the complex. These results were supported by T(2) measurements. Complete resonance assignment of the 11.2 kDa complex of alpha-BTX bound to the alphaAChR peptide comprising residues 182-202 was obtained at pH 4 using homonuclear 2D NMR spectra measured at 800 MHz. The secondary structures of both alpha-BTX and the bound alphaAChR peptide were determined using 2D (1)H NMR experiments. The peptide folds into a beta-hairpin conformation, in which residues (R)H186-(R)V188 and (R)Y198-(R)D200 form the two beta-strands. Residues (R)Y189-(R)T191 form an intermolecular beta-sheet with residues (B)K38-(B)V40 of the second finger of alpha-BTX. These results accurately pinpoint the alpha-BTX-binding site on the alphaAChR and pave the way to structure determination of this important alphaAChR determinant involved in binding acetylcholine and cholinergic agonists and antagonists.

Intramolecular interactions in bound cholera toxin peptide (CTP3) in three antibody complexes were studied by two-dimensional transferred NOE spectroscopy. These measurements together with previously recorded spectra that show intermolecular interactions in these complexes were used to obtain restraints on interproton distances in two of these complexes (TE32 and TE33). The NMR-derived distance restraints were used to dock the peptide into calculated models for the three-dimensional structure of the antibody combining site. It was found that TE32 and TE33 recognize a loop comprising the sequence VPGSQHID and a beta-turn formed by the sequence VPGS. The third antibody, TE34, recognizes a different epitope within the same peptide and a beta-turn formed by the sequence IDSQ. Neither of these two turns was observed in the free peptide. The formation of a beta-turn in the bound peptide gives a compact conformation that maximizes the contact with the antibody and that has greater conformational freedom than alpha-helix or beta-sheet secondary structure. A total of 15 antibody residues are involved in peptide contacts in the TE33 complex, and 73% of the contact area in the antibody combining site consists of the side chains of aromatic amino acids. A comparison of the NMR-derived models for CTP3 interacting with TE32 and TE33 with the previously derived model for TE34 reveals a relationship between amino acid sequence and combining site structure and function. (a) The three aromatic residues that interact with the peptide in TE32 and TE33 complexes, Tyr 32L, Tyr 32H, and Trp 50H, are invariant in all light chains sharing at least 65% identity with TE33 and TE32 and in all heavy chains sharing at least 75% identity with TE33. Although TE34 differs from TE32 and TE33 in its fine specificity, these aromatic residues are conserved in TE34 and interact with its antigen. Therefore, we conclude that the role of these three aromatic residues is to participate in nonspecific hydrophobic interactions with the antigen. (b) Residues 31, 31c, and 31e of CDR1 of the light chain interact with the antigen in all three antibodies that we have studied. The amino acids in these positions in TE34 differ from those in TE32 and TE33, and they are involved in specific polar interactions with the antigen. (c) CDR3 of the heavy chain varies considerably both in length and in sequence between TE34 and the two other anti-CTP3 antibodies. These changes modify the shape of the combining site and the hydrophobic and polar interactions of CDR3 with the peptide antigen.

The TE34 monoclonal antibody against cholera toxin peptide 3 (CTP3; VEVPGSQHIDSQKKA) was sequenced and investigated by two-dimensional transferred NOE difference spectroscopy and molecular modeling. The VH sequence of TE34, which does not bind cholera toxin, shares remarkable homology to that of TE32 and TE33, which are both anti-CTP3 antibodies that bind the toxin. However, due to a shortened heavy chain CDR3, TE34 assumes a radically different combining site structure. The assignment of the combining site interactions to specific peptide residues was completed by use of AcIDSQRKA, a truncated peptide analogue in which lysine-13 was substituted by arginine, specific deuteration of individual polypeptide chains of the antibody, and a computer model for the Fv fragment of TE34. NMR-derived distance restraints were then applied to the calculated model of the Fv to generate a three-dimensional structure of the TE34/CTP3 complex. The combining site was found to be a very hydrophobic cavity composed of seven aromatic residues. Charged residues are found in the periphery of the combining site. The peptide residues HIDSQKKA form a beta-turn inside the combining site. The contact area between the peptide and the TE34 antibody is 388 A2, about half of the contact area observed in protein-antibody complexes.

The interactions between the aromatic amino acids of two monoclonal antibodies (TE32 and TE33) with specific amino acid residues of a peptide of cholera toxin (CTP3) have been determined by two-dimensional (2D) transferred NOE difference spectroscopy. Aromatic amino acids are found to play an important role in peptide binding. In both antibodies two tryptophan and two tyrosine residues and one histidine residue interact with the peptide. In TE33 there is an additional phenylalanine residue that also interacts with the peptide. The residues of the CTP3 peptide that have been found to interact with the antibody are val 3, pro 4, gly 5, gln 7, his 8, and asp 10. We have determined the amino acid sequences of the two antibodies by direct mRNA sequencing. Computerized molecular modeling has been used to build detailed all-atom models of both antibodies from the known conformations of other antibodies. These models allow unambiguous assignment of most of the antibody residues that interact with the peptide. A comparison of the amino acid sequences of the two anti-CTP3 antibodies with other antibodies from the same gene family reveals that the majority of the aromatic residues involved in the binding of CTP3 are conserved although these antibodies have different specificities. This similarity suggests that these aromatic residues create a general hydrophobic pocket and that other residues in the complementarity-determining regions (CDRs) modulate the shape and the polarity of the combining site to fit the specific antigens.