Side-chain Protons Research Articles

RAP Uses a Histidine Switch to Regulate Its Interaction with LRP in the ER and Golgi

The receptor associated protein (RAP) is an antagonist and molecular chaperone that binds tightly to low-density lipoprotein receptor family members in the endoplasmic reticulum (ER). After escorting these receptors to the Golgi, RAP dissociates from the receptors. The molecular mechanism of the dissociation has been unknown until now. The solution structure of RAP-D3 domain presented here reveals a striking increase in positively charged residues on the surface of this RAP domain due to protonation of solvent-exposed histidine sidechains as the pH is reduced from a near neutral pH of the ER to the acidic pH of the Golgi. Structure-based mutagenesis studies in vitro and in cells confirm that the protonation of histidine residues as a consequence of the pH changes modulate the binding/release of RAP from LRP. This histidine switch may serve as a general mechanism for regulating cell trafficking events.

Function and Solution Structure of Huwentoxin-X, a Specific Blocker of N-type Calcium Channels, from the Chinese Bird Spider Ornithoctonus huwena

Huwentoxin-X (HWTX-X) is a novel peptide toxin, purified from the venom of the spider Ornithoctonus huwena. It comprises 28 amino acid residues including six cysteine residues as disulfide bridges linked in the pattern of I-IV, II-V, and III-VI. Its cDNA, determined by rapid amplification of 3' and 5' cDNA ends, encodes a 65-residue prepropeptide. HWTX-X shares low sequence homology with omega-conotoxins GVIA and MVIIA, two well known blockers of N-type Ca2+ channels. Nevertheless, whole cell studies indicate that it can block N-type Ca2+ channels in rat dorsal root ganglion cells (IC50 40 nm) and the blockage by HWTX-X is completely reversible. The rank order of specificity for N-type Ca2+ channels is GVIA approximately HWTX-X > MVIIA. In contrast to GVIA and MVIIA, HWTX-X had no detectable effect on the twitch response of rat vas deferens to low frequency electrical stimulation, indicating that HWTX-X has different selectivity for isoforms of N-type Ca2+ channels, compared with GVIA or MVIIA. A comparison of the structures of HWTX-X and GVIA reveals that they not only adopt a common structural motif (inhibitor cystine knot), but also have a similar functional motif, a binding surface formed by the critical residue Tyr, and several basic residues. However, the dissimilarities of their binding surfaces provide some insights into their different selectivities for isoforms of N-type Ca2+ channels.

Sequence-specific Assignment of <sup>1</sup>H-NMR Resonance and Determination of the Secondary Structure of Jingzhaotoxin-I

Jingzhaotoxin-I (JZTX-I) purified from the venom of the spider Chilobrachys jingzhao is a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3. The structure of this toxin in aqueous solution was investigated using 2-D 1H-NMR techniques. The complete sequence-specific assignments of proton resonance in the 1H-NMR spectra of JZTX-I were obtained by analyzing a series of 2-D spectra, including DQF-COSY, TOCSY and NOESY spectra, in H2O and D2O. All the backbone protons except for Gln4 and more than 95% of the side-chain protons were identified by d alphaN, d alphadelta, d betaN and d NN connectivities in the NOESY spectrum. These studies provide a basis for the further determination of the solution conformation of JZTX-I. Furthermore, the secondary structure of JZTX-I was identified from NMR data. It consists mainly of a short triple-stranded antiparallel beta-sheet with Trp7-Cys9, Phe20-Lys23 and Leu28-Trp31. The characteristics of the secondary structure of JZTX-I are similar to those of huwentoxin-I (HWTX-I) and hainantoxin-IV (HNTX-IV), whose structures in solution have previously been reported.

PH-induced structural change in a sodium/proton antiporter from Methanococcus jannaschii

Na+/H+ antiporters are pH-dependent membrane transport proteins that maintain the homeostasis of H+ and Na+ in living cells. MjNhaP1 from Methanococcus jannaschii, a hyperthermophilic archaeon that grows optimally at 85 degrees C, was cloned and expressed in Escherichia coli. Two-dimensional crystals were obtained from purified protein at pH 4. Electron cryomicroscopy yielded an 8 A projection map. Like the related E. coli antiporter NhaA, MjNhaP1 is a dimer, but otherwise the structures of the two antiporters differ significantly. The map of MjNhaP1 shows elongated densities in the centre of the dimer and a cluster of density peaks on either side of the dimer core, indicative of a bundle of 4-6 membrane-spanning helices. The effect of pH on the structure of MjNhaP1 was studied in situ. A major change in density distribution within the helix bundle, and an approximately 2 A shift in the position of the helix bundle relative to the dimer core occurred at pH 6 and above. The two conformations at low and high pH most likely represent the closed and open states of the antiporter.

Electrostatic stabilization in a pre-organized polar active site: the catalytic role of Lys-80 in Candida tenuis xylose reductase (AKR2B5) probed by site-directed mutagenesis and functional complementation studies

Lys-80 of Candida tenuis xylose reductase (AKR2B5) is conserved throughout the aldo-keto reductase protein superfamily and may prime the nearby Tyr-51 for general acid catalysis to NAD(P)H-dependent carbonyl group reduction. We have examined the catalytic significance of side-chain substitutions in two AKR2B5 mutants, Lys-80-->Ala (K80A) and Asp-46-->Asn Lys-80-->Ala (D46N K80A), using steady-state kinetic analysis and restoration of activity with external amines. Binding of NAD+ (Kd = 24 microM) and NADP+ (Kd = 0.03 microM) was 10- and 40-fold tighter in K80A than the wild-type enzyme, whereas binding of NADH (Kd = 51 microM) and NADPH (Kd = 19 microM) was weakened 2- and 16-fold in this mutant respectively. D46N K80A bound NAD(P)H and NAD(P)+ uniformly approx. 5-fold less tightly than the wild-type enzyme. The second-order rate constant for non-covalent restoration of NADH-dependent reductase activity (kmax/Kamine) by protonated ethylamine was 0.11 M(-1).s(-1) for K80A, whereas no detectable rescue occurred for D46N K80A. After correction for effects of side-chain hydrophobicity, we obtained a linear free energy relationship of log (kmax/Kamine) and amine group pKa (slope = +0.29; r2 = 0.93) at pH 7.0. pH profiles of log (kcat/Km) for carbonyl group reduction by wild-type and D46N K80A revealed identical and kinetically unperturbed pKa values of 8.50 (+/-0.20). Therefore the protonated side chain of Lys-80 is not an essential activator of general acid catalysis by AKR2B5. Stabilized structurally through the salt-link interaction with the negatively charged Asp-46, it is proposed to pull the side chain of Tyr-51 into the catalytic position, leading to a preorganized polar environment of overall neutral charge, in which approximation of uncharged reactive groups is favoured and thus hydride transfer from NAD(P)H is strongly preferred. Lys-80 affects further the directional preference of AKR2B5 for NAD(P)H-dependent reduction by increasing NAD(P)H compared with NAD(P)+-binding selectivity.

The Solution Structure of a Transient Photoreceptor Intermediate: Δ25 Photoactive Yellow Protein

The N-terminally truncated variant of photoactive yellow protein (Delta25-PYP) undergoes a very similar photocycle as the corresponding wild-type protein (WT-PYP), although the lifetime of its light-illuminated (pB) state is much longer. This has allowed determination of the structure of both its dark- (pG) as well as its pB-state in solution by nuclear magnetic resonance (NMR) spectroscopy. The pG structure shows a well-defined fold, similar to WT-PYP and the X-ray structure of the pG state of Delta25-PYP. In the long-lived photocycle intermediate pB, the central beta sheet is still intact, as well as a small part of one alpha helix. The remainder of pB is unfolded and highly flexible, as evidenced by results from proton-deuterium exchange and NMR relaxation studies. Thus, the partially unfolded nature of the presumed signaling state of PYP in solution, as suggested previously, has now been structurally demonstrated.

Chiral discrimination in binding of enantiomers of 2-(aminoalkoxy)-substituted 4-(2-thienyl)pyrimidines and 4,6-bis(2-thienyl)pyrimidines with duplex DNA

Thienylpyrimidines substituted at position 2 of the pyrimidine with a chiral aminoalkoxy group were synthesized. Upon interaction with duplex DNA, the unfused heteroaromatic system of these compounds intercalates with DNA base pairs and the protonated side chain is located in the major groove. The S-enantiomers bind more strongly than their R-counterparts with enantiomeric discrimination, as measured by a ratio of binding constants K S / K R , ranging from 1.2 to 2.4.

High-resolution aliphatic side-chain assignments in 3D HCcoNH experiments with joint H–C evolution and non-uniform sampling

We describe an efficient NMR triple resonance approach that correlates, at high resolution, protein side-chain and backbone resonances. It relies on the combination of two strategies: joint evolution of aliphatic side-chain proton/carbon coherences using a backbone N-H based HCcoNH reduced dimensionality (RD) experiment and non-uniform sampling (NUS) in two indirect dimensions. A typical data set containing such correlation information can be acquired in 2 days, at very high resolution unfeasible for conventional 4D HCcoNH-TOCSY experiments. The resonances of the aliphatic side-chain protons are unambiguously assigned to their attached carbons through the analysis of the 'sum' and 'difference' spectra. This approach circumvents the tedious process of manual resonance assignments using HCcH-TOCSY data, while providing additional resolving power of backbone N-H signals. A simple peak-list based algorithm has been implemented in the IBIS software for rapid automated backbone and side-chain assignments.

Dynamic Mechanism for the Serpin Loop Insertion as Revealed by Quantitative Kinetics

The serpin conformational change by insertion of the reactive center loop into beta-sheet A plays a central role in multiple physiological consequences such as serine proteinase inhibition, latency and serpinopathic polymerization. To study the dynamic mechanism for the loop insertion, a novel kinetic method was established utilizing the ovalbumin mutant R339T/A352R; the loop insertion progressed after the cleavage of P1-P1' (Arg352-Ser353) by trypsin was quenched at pH 8 and 0.5 degrees C, and different conformers were quantified by separation using ion-exchange HPLC. The apparent first-order rate constant k(app) determined for various R339T/A352R derivatives differing in conformational stability was greatly increased by lowering the pH. The pH-dependence of k(app) indicated that the protonation of side-chain(s) with a pK(a) value of around 4.6 is a pre-requisite for the loop insertion. The theoretical rate constant k for the protonated form calculated from k(app) was highly variable, depending on the ovalbumin derivative; structural modifications that give increased mobility to helix F and the sheet-A half (s3A/s2A/s1A) resulted in a striking increase in the loop insertion rate constant k. The k values were determined at different temperatures for all the ovalbumin derivatives, and DeltaH(double dagger) and DeltaS(double dagger) values for the loop insertion reaction were determined according to the transition theory. The formation of the transition state was highly endothermic with minor entropy gain, requiring a DeltaG(double dagger) larger than 18 kcal/mol, which can offset the hydrogen-bond cleavages between s3A and s5A. These results are consistent with the transition state with an opened sheet A and altered orientation of helix F.

Switching of turn conformation in an aspartate anion peptide fragment by NH · · · O− hydrogen bonds

Aspartic acid protease model peptides Z-Phe-Asp(COOH)-Thr-Gly-Ser-Ala-NHCy (1) and AdCO-Asp(COOH)-Val-Gly-NHBzl (3), and their aspartate anions (NEt4)[Z-Phe-Asp(COO-)-Thr-Gly-Ser-Ala-NHCy] (2) and (NEt4)[AdCO-Asp(COO-)-Val-Gly-NHBzl] (4), having an invariant primary sequence of the Asp-X(Thr,Ser)-Gly fragment, were synthesized and characterized by 1H-NMR, CD, and infrared (IR) spectroscopies. NMR structure analyses indicate that the Asp O(delta) atoms of the aspartate peptide 2 are intramolecularly hydrogen-bonded with Gly, Ser, Ala NH, and Ser OH, supporting the rigid beta-turn-like conformation in acetonitrile solution. The tripeptide in the aspartic acid 3 forms an inverse gamma-turn structure, which is converted to a beta-turn-like conformation because of the formation of the intramolecular NH . . . O- hydrogen bonds with the Asp O(delta) in 4. Such a conformational change is not detected between dipeptides AdCO-Asp(COOH)-Va-NHAd (5) and (NEt4)[AdCO-Asp(COO-)-Val-NHAd] (6). The pK(a) value of side-chain carboxylic acid (5.0) for 3 exhibits a lower shift (0.3 unit) from that of 5 in aqueous polyethyleneglycol lauryl ether micellar solution. NMR structure analyses for 3 in an aqueous micellar solution indicate that the preorganized turn structure, which readily forms the NH . . . O- hydrogen bonds, lowers the pK(a) value and that resulting hydrogen bonds stabilize the rigid conformation in the aspartate anion state. We found that the formation of the NH . . . O- hydrogen bonds involved in the hairpin turn is correlated with the protonation and deprotonation state of the Asp side chain in the conserved amino acid fragments.

Solution Structure and Stability against Digestion of rproBnIb, a Recombinant 2S Albumin from Rapeseed: Relationship to Its Allergenic Properties,

NMR spectroscopy has been used to determine the solution structure of the precursor form of the recombinant napin BnIb, rproBnIb, a 2S albumin, 109-residue protein from the seeds of Brassica napus. More than 90% of the side-chain proton resonances were unambiguously assigned from the analysis of two-dimensional correlation (COSY), total correlation (TOCSY), and nuclear Overhauser effect (NOESY) spectra. The final structures were computed by using restrained molecular dynamics on the basis of 1316 upper-limit distance constraints derived from NOE cross-correlation intensities. The computed structures exhibited a root-mean-square deviation (RMSD) radius of 0.66 A for the backbone and 1.16 A for the side-chain heavy atoms of the structural core. The resulting structure consists of five amphipathic helices arranged in a right-handed super helix, a folding motif found in other proteins of the prolamin superfamily. As in the case of the mature protein, the recombinant precursor behaves as a plant food allergen. To trace out the origin and characteristics of its allergenic properties, rproBnIb was assayed against simulated gastric fluid and found to be very resistant to proteolysis. Also, heat treatment of the protein followed up to 85 degrees C by circular dichroism showed a very limited unfolding, which was recovered after cooling to 20 degrees C, indicating a high thermal stability. These results suggest that rproBnIb, as other 2S albumins, may be able to reach the gut immune system intact. A comparison of the putative epitopes against IgE antibodies of the three members of the prolamine family [2S albumins, nonspecific lipid transfer proteins (nsLTPs), and alpha-amylase/trypsin inhibitors] indicates that there are not common surfaces of interaction with IgE. Though the epitopes appear to be located in different regions of the proteins, they do comply with the requirements of being solvent-exposed and flexible.

Nonergodic and conformational control of the electron capture dissociation of protein cations.

Electron capture dissociation (ECD) MS is proving to be unusually valuable for "top down" protein sequencing and identification/localization of posttranslational modifications, because the ECD product ions can represent cleavages between most of a protein's amino acids. As proposed, this unusual reactivity results from immediate local utilization, before randomization, of much of the relatively large ( approximately 6 eV) energy from the electron reaction with the multiply charged protein ion, minimizing the effect of differences in the backbone bond dissociation energies. However, others conclude that e(-) capture produces a labile free radical species for which backbone cleavage is the lowest energy reaction. Supporting the nonergodic mechanism, ECD of ubiquitin (M + 12H)(12+) ions also yields thermalized radical (M + 12H)(11+.) ions that instead lose H. when activated. Also, the ECD spectrum of ubiquitin (M + 13H)(13+) ions is nearly unchanged by heating from 25 degrees C to 125 degrees C, demonstrating that this increase in thermal energy is small compared to the energy driving the reaction. These results support initial capture of the electron in a long-lived high-n Rydberg state, followed by internal conversion to the product valence state at an energy well above the dissociation barriers. The instantaneous conformation of the valence state is critical, with the observed products supporting an alpha-helical structure in which the protonated side chain of each basic residue is intercalated to hydrogen-bond to as many as three amide carbonyls. Activation (e.g., heat, collisions, lowered charge) can disrupt this conformation to allow additional H(+)-side-chain interactions and provide more complete sequence coverage.

Solution structure of the human oncogenic protein gankyrin containing seven ankyrin repeats and analysis of its structure--function relationship.

Human gankyrin (226 residues, 24.4 kDa) is a liver oncoprotein that plays an important role in the development of human hepatocellular carcinomas. In this paper, its solution structure is reported, which is the largest ankyrin protein ever determined by NMR. The highly degenerate primary sequences of the seven ankyrin repeats presented a major challenge, which was overcome by combined use of TROSY experiments, perdeuterated samples, isotope-filtered NMR experiments, and residual dipolar couplings. The final structure was of high quality, with atomic rmsds for the backbone (N, C', and C(alpha)) and all heavy atoms (residues 4-224) of 0.69 +/- 0.09 and 1.04 +/- 0.09 A, respectively. Detailed analyses of NMR data suggested that the conserved TPLH motifs play important structural roles in stabilizing the repeating ankyrin scaffold. Gankyrin is conformationally more stable than the tumor suppressor p16(INK4A), possibly due to the structural roles of conserved residues evidenced by slowly exchanging backbone amides as well as hydrogen bonding networks involving labile side chain protons. Structural comparison with p16(INK4A) identified several residues of gankyrin that are potentially important for CDK4 binding, whereas observation of the thiol proton of C180 indicated a well-structured Rb-binding site in the helical region of the sixth ankyrin repeat. Interestingly, the CDK4-binding site and Rb-binding site located in N- and C-terminal regions, respectively, are separated by comparatively more stable ankyrin repeats and highly condensed positive surface charge. These results and analyses will shed light on the structural basis of the function of human gankyrin.

Side chain-side chain interactions of arginine with tyrosine and aspartic acid in Arg/Gly/Tyr-rich domains within plant glycine-rich RNA binding proteins.

Plant glycine-rich RNA-binding proteins (GRRBPs) contain a glycine-rich region at the C-terminus whose structure is quite unknown. The C-terminal glycine-rich part is interposed with arginine and tyrosine (arginine/glycine/tyrosine (RGY)-rich domain). Comparative sequence analysis of forty-one GRRBPs revealed that the RGY-rich domain contains multiple repeats of Tyr-(Xaa)h-(Arg)k-(Xaa)l, where Xaa is mainly Gly, "k" is 1 or 2, and "h" and "l" range from 0 to 10. Two peptides, 1 (G1G2Y3G4G5G6R7R8D9G10) and 2 (G1G2R3R4D5G6G7Y8G9G10), corresponding to sections of the RGY-rich domain in Zea mays RAB15, were selected for CD and NMR experiments. The CD spectra indicate a unique, positive band near 228 nm in both peptides that has been ascribed to tyrosine residues in ordered structures. The pH titration by NMR revealed that a side chain-side chain interaction, presumably an H-Nepsilon...O=Cgamma hydrogen bonding interaction in the salt bridge, occurs between Arg (i) and Asp (i + 2). 1D GOESY experiments indicated the presence of NOE between the aromatic side chain proton and the arginine side chain proton in the two peptides suggesting strongly that the Arg (i) aromatic side chain interacts directly with the Tyr (i +/- 4 or i +/- 5) side chain.

Theoretical Investigations on Azotobacter vinelandii Ferredoxin I: Effects of Electron Transfer on Protein Dynamics

Structural, energetic, and dynamical studies of Azotobacter vinelandii ferredoxin I are presented for native and mutant forms. The protein contains two iron-sulfur clusters, one of which ([3Fe-4S]) is believed to play a central role in the electron-coupled proton transfer. Different charge sets for the [3Fe-4S] cluster in its reduced and oxidized state are calculated with broken symmetry ab initio density functional theory methods and used in molecular dynamics (MD) simulations. The validity of the ab initio calculations is assessed by comparing partially optimized structures of the [3Fe-4S] clusters with x-ray structures. Possible proton transfer pathways between the protein and the iron-sulfur cluster are examined by both MD simulations and ab initio calculations. The MD simulations identify three main-chain hydrogen atoms—HN(13), HN(14), and HN(16)—that are within H-bonding distance of the [3Fe-4S] cluster throughout the MD simulations. They could thus play a role in the proton transfer from the protein to the iron-sulfur cluster. By contrast, the HD2(15) atom of the Asp-15 is seldom close enough to the [3Fe-4S] cluster to transfer a proton. Poisson-Boltzmann calculations indicate that there is a low, but nonzero probability, that Asp-15 is protonated at pH 7; this is a requirement for it to serve as a proton donor. Ab initio calculations with a fragment model for the protein find similar behavior for the transfer of a proton from the OH of the protonated side chain and the main-chain NH of Asp-15. The existence of a stable salt bridge between Asp-15 and Lys-84 in the D15E mutant, versus its absence in the wild-type, has been suggested as the cause of the difference in the rate of proton transfer. Extensive MD simulations were done to test this idea; the results do not support the proposal. The present findings, together with the available data, serve as the basis for an alternative proposal for the mechanism of the coupled electron-proton transfer reaction in ferredoxin I.

Improved method for unambiguous amino acid side-chain 1H and 13C resonance assignment.

Side-chain proton and carbon-13 resonance assignments of [13C;15N]-enriched proteins usually rely on combinations of several multi-dimensional experiments. Here, we describe a four-dimensional pulse sequence, H(C)C-COSY-TOCSY-(CACO)NH, which provides the information required to assign completely aliphatic side-chain resonance frequencies. As in widely used HCC(CO)NH-TOCSY experiments, problems due to spectral crowding are alleviated by exploiting the dispersion of backbone amide 1H and 15N signals. The modification introduced here allows signals from different side-chains to be distinguished even in the case of overlap in the 1H(N)-15N plane of the spectra. For illustration, the new method is applied to two proteins with molecular masses of 11 and 23 kDa.

Copper binding to plant ozone-inducible proteins (OI2-2 and OI14-3)

Ozone-inducible proteins (OI2-2 and OI14-3) from Atriplex canescens whose structure and function are unknown are rich in glycine intercepted with histidine and tyrosine with putative signal peptides at the N-terminus. OI2-2 and OI14-3 contain 8 and 10 tandem repeats of YGHGGG, respectively. In order to study whether these proteins bind Cu 2+, circular dichroism (CD), and nuclear magnetic resonance (NMR) were measured for four synthetic peptides corresponding to sections of the sequences of these proteins; 1 (HGGGY), 2 (HGGGYGH), 3 (YGHGGGY), and 4 (YGHGGGYGHGGGY), where all peptides were chemically blocked with an acetyl group at the N-terminus and an –NH 2 group at the C-terminus. Visible CD spectra of the four peptides show positive peaks near 580 and 340 nm, which were observed at pH 7.4 but not pH 6.0, indicating clearly that the four peptides bind Cu 2+. The NMR spectra indicate that the addition of small amounts of CuSO 4 to 3 (Y1–G2–H3–G4–G5–G6–Y7) causes significant broadening of resonances of the side chain protons (C βH, C ε1H, and C δ2H) of His3 and the side chain C βH of Tyr1 at pH 7.4. In addition, the backbone C αH resonances of Gly2 and Gly4 were broadened more strongly than those of Gly5 and Gly6. CD titration experiment suggested that two repeats of YGHGGG comprise the fundamental Cu 2+ binding unit. Thus, the ozone-inducible proteins are capable of binding at least four or five copper ions per protein. These copper-binding proteins would function as active oxygen scavengers.

Structural basis of ligand recognition by PABC, a highly specific peptide-binding domain found in poly(A)-binding protein and a HECT ubiquitin ligase.

The C-terminal domain of poly(A)-binding protein (PABC) is a peptide-binding domain found in poly(A)-binding proteins (PABPs) and a HECT (homologous to E6-AP C-terminus) family E3 ubiquitin ligase. In protein synthesis, the PABC domain of PABP functions to recruit several translation factors possessing the PABP-interacting motif 2 (PAM2) to the mRNA poly(A) tail. We have determined the solution structure of the human PABC domain in complex with two peptides from PABP-interacting protein-1 (Paip1) and Paip2. The structures show a novel mode of peptide recognition, in which the peptide binds as a pair of beta-turns with extensive hydrophobic, electrostatic and aromatic stacking interactions. Mutagenesis of PABC and peptide residues was used to identify key protein-peptide interactions and quantified by isothermal calorimetry, surface plasmon resonance and GST pull-down assays. The results provide insight into the specificity of PABC in mediating PABP-protein interactions.

Biomimetic approaches to gas phase peptide chemistry: combining selective binding motifs with reactive carbene precursors to form molecular mousetraps

Biomimetic reagents capable of selectively forming non-covalent complexes and initiating intermolecular reactions with peptides in the gas phase are presented. In the present work, 18-crown-6 ether (18C6) is utilized to bind specifically to various protonated primary amines, including the protonated side chain of lysine. The use of multiple crown ethers is shown to be an efficient method for enhancing the binding energy, which is a critical factor influencing the success of these reagents. The binding energy must exceed any reaction barriers to the desired chemistry, otherwise simple dissociation of the complex occurs. Two reagents containing acidic and transition metal binding functionalities, respectively, designed to selectively cleave peptide bonds, are synthesized and tested experimentally. A third class of reagent designed to covalently attach to peptides utilizing carbene insertion chemistry is also presented. The results demonstrate that combining the recognition and binding powers of 18C6 with an easily activated diazo group allows for the efficient generation of a highly reactive carbene within a non-covalent complex. Intermolecular insertion reactions initiated by the carbene can transform these non-covalent complexes into covalently bound molecules. Electrospray ionization mass spectrometry and density functional theory (DFT) are utilized to evaluate these intermolecular insertion reactions. The results from experiments with several small molecules and peptides are presented. These diazo-based reagents prove to be highly versatile molecules capable of binding to, and with appropriate activation, becoming covalently attached to virtually any molecule that contains a primary amine. For this reason, we have dubbed them “molecular mousetraps.”

Direct observation of protonation reactions during the catalytic cycle of cytochrome c oxidase.

Cytochrome c oxidase, the terminal protein in the respiratory chain, converts oxygen into water and helps generate the electrochemical gradient used in the synthesis of ATP. The catalytic action of cytochrome c oxidase involves electron transfer, proton transfer, and O2 reduction. These events trigger specific molecular changes at the active site, which, in turn, influence changes throughout the protein, including alterations of amino acid side chain orientations, hydrogen bond patterns, and protonation states. We have used IR difference spectroscopy to investigate such modulations for the functional intermediate states E, R2,Pm, and F. These spectra reveal deprotonation of its key glutamic acid E286 in the E and in the Pm states. The consecutive deprotonation and reprotonation of E286 twice within one catalytic turnover illustrates the role of this residue as a proton shuttle. In addition, the spectra point toward deprotonation of a redox-active tyrosine, plausibly Y288, in the F intermediate. Structural insights into the molecular mechanism of catalysis based on the subtle molecular changes observed with IR difference spectroscopy are discussed.