Periodicity of amide proton exchange rates in a coiled-coil leucine zipper peptide.

The two-stranded coiled-coil motif, which includes leucine zippers, is a simple protein structure that is well suited for studies of helix-helix interactions. The interaction between helices in a coiled coil involves packing of "knobs" into "holes", as predicted by Crick in 1953 and confirmed recently by X-ray crystallography for the GCN4 leucine zipper [O'Shea, E.K., Klemm, J.D., Kim, P.S., & Alber, T. (1991) Science 254, 539]. A striking periodicity, extending over six helical turns, is observed in the rates of hydrogen-deuterium exchange for amide protons in a peptide corresponding to the leucine zipper of GCN4. Protons at the hydrophobic interface show the most protection from exchange. The NMR chemical shifts of amide protons in the helices also show a pronounced periodicity which predicts a short H-bond followed by a long H-bond every seven residues. This variation was anticipated in 1953 by Pauling and is sufficient to give rise to a local left-handed superhelical twist characteristic of coiled coils. The amide protons that lie at the base of the "hole" in the "knobs-into-holes" packing show slow amide proton exchange rates and are predicted to have short H-bond lengths. These results suggest that tertiary interactions can lead to highly localized, but substantial, differences in stability and dynamics within a secondary structure element and emphasize the dominant nature of packing interactions in determining protein structure.     

Protection of amide protons in folding intermediates of ribonuclease A measured by pH-pulse exchange curves

pH-pulse exchange curves have been measured for samples taken during the folding of ribonuclease A. The curve gives the number of protected amide protons remaining after a 10-s pulse of exchange at pHs from 6.0 to 9.5, at 10 degrees C. Amide proton exchange is base catalyzed, and the rate of exchange increases 3000-fold between pH 6.0 and pH 9.5. The pH at which exchange occurs depends on the degree of protection against exchange provided by structure. Pulse exchange curves have been measured for samples taken at three times during folding, and these are compared to the pulse exchange curves of N, the native protein, of U, the unfolded protein in 4 M guanidinium chloride, and of IN, the native-like intermediate obtained by the prefolding method of Schmid. The results are used to determine whether folding intermediates are present that can be distinguished from N and U and to measure the average degree of protection of the protected protons in folding intermediates. The amide (peptide NH) protons of unfolded ribonuclease A were prelabeled with 3H by a previous procedure that labels only the slow-folding species. Folding was initiated at pH 4.0, 10 degrees C, where amide proton exchange is slower than the folding of the slow-folding species. Samples were taken at 0-, 10-, and 20-s folding, and their pH-pulse exchange curves were measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Utilization of selected leucine peptide amides by Escherichia coli.

Studies on the utilization of leucine peptide amides as a source of leucine for a leucine auxotroph showed that in general compounds with the structure leu-chi amide (where chi is any amide) are utilized as well as the free peptide, but that compounds with the structure chi-leu amide (where chi is not leucine) are used less effectively than the free peptide. Growth and enzymological experiments indicated that the lower capacity of Escherichia coli to utilize amides of the structure chi-leu amide is not a result of poor transport of these compounds, but rather the inability to rapidly liberate leucine from the amide when it is supplied to the cell in the form of a peptide. Competition studies indicated that the peptide amides enter the cell via the oligopeptide permease system.     

Characterization of a proteolipid complex of aminoacyl-tRNA synthetases and transfer RNA from rat liver.

A high molecular weight complex containing aminoacyl-tRNA synthetases, peptidyl acetyltransferase, lipids and tRNA has been isolated from the 250,000 x g postmitochondrial supernatant from rat liver cells. Aminoacyl-tRNA synthetase activity directed towards arginine, aspartate, glutamine, glutamate, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, and tyrosine is present. An endogenous pool of aminoacyladenylates is indicated by an ATP-32PPi exchange catalyzed by the native complex, which shows a dramatic increase after addition of ATP. Lysine is the only amino acid which greatly increases the exchange rate catalyzed by the native complex in vitro, whereas components of the denatured complex activate all the 13 amino acids in the presence of ATP. Six of the eight lipid fractions were glycolipids; cholesterol and cholesterol esters were absent. The extracted RNA has many characteristics of tRNA. These findings provide evidence for the organization of aminoacyl-tRNA synthetases in a complex with peptidyl acetyltransferase that also contains lipids and tRNA and that can be readily isolated from the cytosol of rat liver cells.     

A hydrogen-deuterium exchange study of the amide protons of polymyxin B by nuclear-magnetic-resonance spectroscopy.

1. Proton magnetic resonance spectra at 270 MHz of polymyxin B, a cationic oligopeptide antibiotic, show the influence of the inorganic counteranion present in solution. 2. Hydrogen-deuterium exchange rates for the amide protons are of two types, depending on whether the anion is monovalent or polyvalent. Polyvalent anions catalyse the acid-catalysed reaction more than the monovalent anions. 3. The structure in solution was monitored using the proton signals of the amides, the phenylalanine aromatic protons, and the leucine methyl and gamma-CH protons in several polymyxin salts. The temperature coefficients of the chemical shifts of the N-H protons are used to identify two beta turns in the cyclic ring of polymyxin B. The variation in chemical shift of the N-H protons, the aromatic protons and the leucine protons are correlated with anionic size and electronegativity.     

Mechanistic studies of amide bond scission during acidolytic deprotection of Pip containing peptide.

Unusual TFA catalyzed cleavage reaction is reported for peptide containing pipecolic acid residues. Although the use of TFA under standard cleavage conditions is sufficiently mild to prevent degradation of the desired products, the amide bond between consecutive pipecolic acid residues is unexpectedly hydrolyzed by standard TFA treatment. The hydrolysis is proposed to proceed via an oxazolinium ion intermediate. This mechanism is supported by H/D exchange as observed by ESI-MS and NMR experiments. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Enzymatic amidation of recombinant (Leu27) growth hormone releasing hormone-Gly45

By chemoenzymatic synthesis the gene for a (Leu27) analogue of human growth hormone releasing hormone-Gly45 [(Leu27)GHRH-Gly45] was constructed, cloned and expressed in Escherichia coli as a fusion protein with beta-galactosidase under the control of the lac promoter and operator. Upon induction with isopropyl-D-thio-beta-galactopyranoside the fusion protein accumulated to a yield of 15-20% of the total cellular protein. After cyanogen bromide cleavage of the fusion protein the precursor peptide (Leu27)hGHRH-Gly45 was separated by extraction and purified by ion exchange and h.p.l.c.-RP18 chromatography. The purified peptide was analysed by sequencing, isoelectric focusing, amino acid analysis and amino acid analysis after V8 protease digestion. The carboxy-terminal glycine was subsequently amidated by PAM (peptidylglycine-alpha-amidating-monooxygenase), an enzyme which was isolated and characterized from fresh bovine pituitaries. Correct amidation of the penultimate amino acid, leucine, was verified by peptide sequencing with an authentic leucine amide reference.     

pH dependency of the carboxyl oxygen exchange reaction catalyzed by lysyl endopeptidase and trypsin

The pH dependency of the carboxyl oxygen exchange reaction catalyzed by lysyl endopeptidase (Lys-C) and trypsin has been studied. The reaction was quantitatively monitored by measuring the incorporation of 18O atom into the alpha-carboxyl group of N(alpha)-acetyl-L-lysine from H2(18)O solvent. The optimum pHs of the carboxyl oxygen exchange reaction catalyzed by Lys-C and trypsin were found to be pH 5.0 and 6.0, respectively, which were significantly shifted toward acidic pHs compared to the most favorable pHs of their amidase activities for N(alpha)-acetyl-L-lysine amide in the pHs examined. Steady-state kinetics parameters were also determined for both enzymes at two different pHs, one at the pH optimum for their carboxyl oxygen exchange activity (pH 5-6) and the other at the favorable pH for their amidase activity (pH 8-9). Significantly lower Km (2-fold lower for Lys-C, 3-fold lower for trypsin), and higher kcat values (1.5-fold higher for Lys-C, 5-fold higher for trypsin) were obtained at the acidic pHs compared to the alkaline pHs, suggesting that Lys-C and trypsin have higher substrate binding affinities and higher catalytic rates at the acidic pHs than at the alkaline pHs. The higher carboxyl oxygen exchange activities at the acidic pHs were also confirmed with peptide substrates derived from apomyoglobin. These findings are significant toward the goal of improving the efficiency of the Lys-C and trypsin catalyzed 18O labeling reactions and are thus pertinent to improving the accuracy and reliability of quantitative proteomic experiments utilizing 18O labeling.     

Solvent Exchange Rates of Side-chain Amide Protons in Proteins

Solvent exchange rates and temperature coefficients for Asn/Gln side-chain amide protons have been measured in Escherichia coli HPr. The protons of the eight side-chain amide groups (two Asn and six Gln) exhibit varying exchange rates which are slower than some of the fast exchanging backbone amide protons. Differences in exchange rates of the E and Z protons of the same side-chain amide group are obtained by measuring exchange rates at pH values > 8. An NOE between a side-chain amide proton and a bound water molecule was also observed.     

Amide hydrogen exchange rates of peptides in H2O solution by 1H nuclear magnetic resonance transfer of solvent saturation method. Conformations of oxytocin and lysine vasopressin in aqueous solution.

The NH exchange rates in aqueous media of oxytocin and 8-lysine vasopressin (LVP) have been measured by using transfer of solvent saturation method. The data are consistent with a "highly motile" dynamic equilibrium between folded and highly solvated conformations. The highly-motility limit applies to the exchange of NH hydrogens of oxytocin and LVP. Folded structures are more prevalent in oxytocin than in LVP. Partial shielding is indicated for peptide hydrogens of Asn5 and perhaps also Cys6 of oxytocin and for Cys6 of LVP. It is tentatively proposed that the folded conformation of oxytocin in aqueous media may contain a parallel beta-structure in the tocinamide ring consisting of two hydrogen bonds: one between the Tyr2 C = O and Asn5 peptide NH as originally proposed for the preferred conformation of oxytocin in dimethyl sulfoxide (D. W. Urry and R. Walter), and the second between he Cys1 C = O and the Cys6 NH. In LVP the hydrogen bond between the Tyr2 C = O and Asn5 peptide NH appears to be absent. The acylic tripeptide sequences (-Pro-X-Gly-NH2) of both hormones appear to be predominantly solvated. The second-order rate constants for acid catalyzed exchange of the primary amide hydrogens of Gln4, Asn5, and Gly9 of oxytocin are consistently greater for the trans NH than for the corresponding cis NH. This observation can be rationalized in terms of mechanisms involving protonation of either the amide oxygen, or the amide nitrogen, but with limited rotation about the C - N bond.     

Separation of rat muscle aminopeptidases.

By means of chromatography on DEAE-Sephadex, two arylamidases (hydrolysing L-arginine 2-naphthylamide) and three dipeptidyl peptidases (hydrolysing dipeptide 2-naphthylamides) were distinguished in extracts of rat muscle. However, the arylamidase from the larger peak also hydrolysed the dipeptide 2-naphthylamides. Glycyl-L-arginine amide, an alternative substrate for dipeptidyl peptidase I, was not hydrolysed by arylamidase. L-Leucine amide was hydrolysed by an enzyme, presumed to be leucine aminopeptidase, from a separate peak, but was also hydrolysed by arylamidase. Arylamidase, dipeptidyl peptidase III and most of the leucine aminopeptidase could be extracted from the muscle with a neutral salt solution, but dipeptidyl peptidase I was extracted only in the presence of Triton X-100; dipeptidyl peptidase II showed an intermediate extraction behaviour.     

TmDOTA-tetraglycinate encapsulated liposomes as pH-sensitive LipoCEST agents

Lanthanide DOTA-tetraglycinate (LnDOTA-(gly)₄ ⁻) complexes contain four magnetically equivalent amide protons that exchange with protons of bulk water. The rate of this base catalyzed exchange process has been measured using chemical exchange saturation transfer (CEST) NMR techniques as a function of solution pH for various paramagnetic LnDOTA-(gly)₄ ⁻ complexes to evaluate the effects of lanthanide ion size on this process. Complexes with Tb(III), Dy(III), Tm(III) and Yb(III) were chosen because these ions induce large hyperfine shifts in all ligand protons, including the exchanging amide protons. The magnitude of the amide proton CEST exchange signal differed for the four paramagnetic complexes in order, Yb>Tm>Tb>Dy. Although the Dy(III) complex showed the largest hyperfine shift as expected, the combination of favorable chemical shift and amide proton CEST linewidth in the Tm(III) complex was deemed most favorable for future in vivo applications where tissue magnetization effects can interfere. TmDOTA-(gly)₄ ⁻ at various concentrations was encapsulated in the core interior of liposomes to yield lipoCEST particles for molecular imaging. The resulting nanoparticles showed less than 1% leakage of the agent from the interior over a range of temperatures and pH. The pH versus amide proton CEST curves differed for the free versus encapsulated agents over the acidic pH regions, consistent with a lower proton permeability across the liposomal bilayer for the encapsulated agent. Nevertheless, the resulting lipoCEST nanoparticles amplify the CEST sensitivity by a factor of ∼10⁴ compared to the free, un-encapsulated agent. Such pH sensitive nano-probes could prove useful for pH mapping of liposomes targeted to tumors.     

Increased stability of peptidesulfonamide peptidomimetics towards protease catalyzed degradation.

Replacement of amide bonds in peptides by sulfonamide moieties resulted in peptidosulfonamides with an increased stability towards protease catalyzed degradation. In addition to protection of the protease cleavage site, it was found that introduction of a sulfonamide also influenced the stability of adjacent amide bonds.     

Individual amide proton exchange rates in thermally unfolded basic pancreatic trypsin inhibitor

A novel experiment is described for measurements of amide proton exchange rates in proteins with a time resolution of about 1 s. A flow apparatus was used to expose protein solutions in 2H2O first to high temperature for a predetermined time period, during which 1H-2H exchange proceeded, and then to ice-water. The technique was applied for exchange studies in thermally unfolded, selectively reduced basic pancreatic trypsin inhibitor. Measurements were made by 1H nuclear magnetic resonance after the exchange was quenched by rapid cooling. Thereby, the sequence-specific resonance assignments for the folded protein could be used, which had been previously obtained. The results of this study indicate that the exchange rates in the thermally unfolded protein are close to those expected for a random chain and that the NH exchange is catalyzed by 2H+ and O2H- up to high temperature, with no significant contributions from p2H-independent catalysis. We conclude that the parameters derived by Molday et al. [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158] from measurements with small model peptides can be used to calculate intrinsic exchange rates in unfolded proteins and thus provide a reliable reference for the interpretation of exchange rates measured under native conditions.     

GuHCl and NaCl-dependent hydrogen exchange in MerP reveals a well-defined core with an unusual exchange pattern

We have analysed hydrogen exchange at amide groups to characterise the energy landscape of the 72 amino acid residue protein MerP. From the guanidine hydrochloride (GuHCl) dependence of exchange in the pre-transitional region we have determined free energy values of exchange (DeltaG(HX)) and corresponding m-values for individual amide protons. Detailed analysis of the exchange patterns indicates that for one set of amide protons there is a weak dependence on denaturant, indicating that the exchange is dominated by local fluctuations. For another set of amide protons a linear, but much stronger, denaturant dependence is observed. Notably, the plots of free energy of exchange versus [GuHCl] for 16 amide protons show pronounced upward curvature, and a close inspection of the structure shows that these residues form a well-defined core in the protein. The hydrogen exchange that was measured at various concentrations of NaCl shows an apparent selective stabilisation of this core. Detailed analysis of this exchange pattern indicates that it may originate from selective destabilisation of the unfolded state by guanidinium ions and/or selective stabilisation of the core in the native state by chloride ions.     

Protein folding kinetics by combined use of rapid mixing techniques and NMR observation of individual amide protons

A method to be used for experimental studies of protein folding introduced by Schmid and Baldwin (J. Mol. Biol. 135: 199-215, 1979), which is based on the competition between amide hydrogen exchange and protein refolding, was extended by using rapid mixing techniques and 1H NMR to provide site-resolved kinetic information on the early phases of protein structure acquisition. In this method, a protonated solution of the unfolded protein is rapidly mixed with a deuterated buffer solution at conditions assuring protein refolding in the mixture. This simultaneously initiates the exchange of unprotected amide protons with solvent deuterium and the refolding of protein segments which can protect amide groups from further exchange. After variable reaction times the amide proton exchange is quenched while folding to the native form continues to completion. By using 1H NMR, the extent of exchange at individual amide sites is then measured in the refolded protein. Competition experiments at variable reaction times or variable pH indicate the time at which each amide group is protected in the refolding process. This technique was applied to the basic pancreatic trypsin inhibitor, for which sequence-specific assignments of the amide proton NMR lines had previously been obtained. For eight individual amide protons located in the beta-sheet and the C-terminal alpha-helix of this protein, apparent refolding rates in the range from 15 s-1 to 60 s-1 were observed. These rates are on the time scale of the fast folding phase observed with optical probes.     

Conformational preference of Leu side chain in melanostatin in DMSO

The solution conformation of melanostatin (Pro-Leu-Gly-NH2) in the neutral and protonated forms of DMSO has been monitored by one and two dimensional NMR techniques at 500 MHz. The temperature coefficients of the amide proton chemical shifts in conjunction with the observed NOESY spectra suggest that melanostatin in neutral form in DMSO adopts a backbone conformation such that leucine amide proton is buried by the proline ring and the side chain of leucine. Similar observation is made for protonated form of melanostatin in DMSO. The results of the present study are at variance with the earlier NMR studies which proposed a beta-turn structure for both the forms of melanostatin. There is, however, no evidence for the presence of beta-turn structure for both the forms of melanostatin in DMSO. In CDCl3 also Leu NH appears to be buried as evident from the solvent titration with DMSO and NOESY spectra.     

pH dependence of hydrogen exchange from backbone peptide amides in apamin

The kinetics of hydrogen exchange of the 11 most protected backbone amides of bee venom apamin have been measured between pH 1 and pH 8.5 by using time-resolved and saturation-transfer NMR spectroscopy. The five amides most protected from base-catalyzed exchange, those of residues 5 and 12-15, show highly correlated exchange behavior in the base-catalyzed regime. It is proposed that the intramolecular hydrogen bonds stabilizing these amides define a stable cooperative unit of secondary structure in apamin (a C-terminal helix and an N-terminal beta-turn). This conformational unit is further stabilized (by 5-6 kJ mol-1) on titration of the Glu-7 side-chain carboxyl group. The relative contributions of specific intramolecular interactions to this conformational stabilization are estimated. The pHminima in the pH-dependent single amide exchange curves are compared with values predicted by correcting for sequence-dependent contributions to amide exchange rates [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158]. The lack of correlation suggests that the "open" conformers from which amide exchange occurs are nonrandom. This conclusion is dependent on the assumption that acid-catalyzed exchange occurs via N-protonation so that residual conformational effects on exchange rates in the open conformers will affect acid- and base-catalyzed rates in approximately equal and opposite ways. A strong correlation between the measured pHminima and the amide proton chemical shifts is observed, however, and this may be most easily accommodated if acid-catalyzed exchange occurs by the imidic acid mechanism (via amide O-protonation).     

Kinetics of amide proton exchange in parvalbumin studied by 1H 2-D NMR. A comparison of the calcium and magnesium loaded forms.

The amide proton exchange rates have been measured for the pike parvalbumin loaded either with calcium (PaCa2) or with magnesium (PaMg2) by using 2-D total correlation spectroscopy experiments. The differences in the exchange rates observed between these two species were unexpected when compared with the small conformational changes induced in parvalbumin by the Ca/Mg exchange. With the calcium-loaded protein (PaCa2), a significant difference was observed for the amide proton exchange rates of residues located in the N-terminal domain AB in contrast to the slower exchange rates that were observed in the CD and EF domains. Such a difference does not exist for PaMg2, where faster exchange rates are observed over all the sequence. Since amide proton exchange rates are the signature of the solvent's accessibility in proteins, we interpreted our results in terms of difference of the equilibria between 'closed-states' and 'opened-states' for individual amide protons of the protein when calcium was replaced by magnesium. The CD and EF domains, and to a lesser extent the AB domain, would be more rigid when the protein was loaded with calcium ions. For the magnesium-loaded parvalbumin (PaMg2) the faster exchange rates we observed could be rationalized by a more flexible structure than in the case of the PaCa2.