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MP2, DFT, and molecular mechanics (AMBER, CVFF, and CFF91) geometry optimizations were performed on the cyclic dipeptide cyclo(L-Pro-L-Pro) starting from crystal structure data. Three stable conformations were identified as energy minima by all methods, but assignment of relative energy varied between the methods. The pi-pi transition feature of the UV circular dichroic (CD) spectrum was predicted for each minimized structure using the classical physics method of the dipole interaction model. The model was sensitive to the different conformations. The UV-CD predictions were compared individually and as a Boltzmann-weighted composite with published experimental CD spectra [Bowman, R. L.; Kellerman, M.; Johnson, W. C., Jr. Biopolymers 1983, 22, 1045]. For all structures, the original parameters of Applequist [Applequist, J. J. Chem. Phys. 1979, 71, 4324] with a bandwidth of 3000 cm(-1) most accurately replicated experiment, except for the CFF91 structures, which matched experiment best with a bandwidth of 4000 cm(-1). The inclusion of solvent by a continuum model did not significantly alter the minimized geometries obtained by molecular or quantum mechanics, but it did have an effect on the relative predicted energies of CFF91 and B3LYP conformations. The overall effect of solvent inclusion was negligible when Boltzmann-weighted spectra were considered. Gas-phase CFF91 structures were also reasonably good for prediction of CD spectra, and when water was included via a continuum model for energy calculations, the weighting scheme resembled that of the higher-level weightings. The CD calculated using the MP2/6-311G structures and energies for weighting were most descriptive of the 180 nm negative band in the experimental CD, but red-shifted the location of the 205 nm band. DFT structures were comparably, though not identically, as descriptive of the first pi-pi band, and did a better job with placement of the second (positive) pi-pi band. DFT calculations were less sensitive to basis set effect than the MP2 calculations, with 6-31G results in close agreement with 6-311G. The results suggest that it is possible to use geometries obtained from a variety of different methods (molecular mechanical or quantum mechanical) with the classical physics dipole interaction model to qualitatively reproduce the UV CD of model amides.
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Rates of collagen and non-collagen protein synthesis in rabbit arterial smooth muscle cells (SMC) were determined by using the specific (radio)activity of [3H]proline in the extracellular, intracellular, and prolyl-tRNA pools. The intracellular free proline specific activity was only 25% of the extracellular value in cultures incubated for 12 h in 0.25 mM-proline. The specific activity of prolyl-tRNA was less than 10% of the extracellular specific activity. Increasing the extracellular proline concentration 10-fold (to 2.5 mM), while keeping the extracellular specific activity of proline constant, resulted in equilibration of the specific activities of intracellular and extracellular free proline, but the specific activity of prolyl-tRNA remained at less than 10% of the extracellular specific activity. Therefore, calculated rates of collagen and non-collagen protein synthesis were greatly underestimated using the intracellular or extracellular specific activity of proline. SMC were also incubated with 0.1 mM-[14C]ornithine in 0.25 nM or 2.5 mM non-labelled proline to examine synthesis de novo of proline and prolyl-tRNA from ornithine. In SMC cultures containing 0.25 mM unlabelled proline, the specific activity of intracellular ornithine was approx. 45% of the extracellular specific activity, due to the production of unlabelled ornithine. The specific activity of ornithine-derived intracellular free proline in SMC incubated with 2.5 mM-proline was significantly lower than in SMC incubated in 0.25 mM-proline, due to the influx of unlabelled proline. However, a corresponding difference in the specific activity of [14C]prolyl-tRNA between SMC in 0.25 mM- or 2.5 mM-proline was not observed. Ornithine-derived [14C]proline was incorporated into proteins in a manner different from that of exogenously added radiolabelled proline. A much higher proportion of the proline synthesized de novo was channelled into collagen synthesis relative to total protein synthesis. Together, these results show that intracellular proline pools are highly compartmentalized in arterial SMC. They also suggest that proline synthesized from ornithine may enter a prolyl-tRNA pool separate from that of proline entering from the extracellular medium.
Several proline derivatives such as L-azetidine-2-carboxylic acid, cis-4-hydroxy-L-proline, and 3,4-dehydro-DL-proline prevent procollagen from folding into a stable triple-helical conformation, thereby reducing excessive deposition of collagen in fibrotic processes and the growth of tumors. This study was performed to investigate whether the recently discovered human proton-coupled amino acid transporter 1 (hPAT1) is capable of transporting such pharmacologically relevant proline derivatives and also GABA analogs. Uptake of L-[3H]proline and [3H]glycine in Caco-2 cells was Na+-independent but strongly H+-dependent. The L-proline uptake was saturable and mediated by a single transport system (hPAT1) with an affinity constant of 2.0 +/- 0.2 mM. The uptake of L-[3H]proline was inhibited by D-proline, trans-4-hydroxy-L-proline, cis-4-hydroxy-L-proline, cis-4-hydroxy-D-proline, 3,4-dehydro-DL-proline, L-azetidine-2-carboxylic acid, 3-amino-1-propanesulfonic acid, D- and L-pipecolic acid, l-thiaproline, and many others. Apical uptake and transepithelial flux of L-[3H]proline across Caco-2 cell monolayers were strongly inhibited by proline derivatives in proportions corresponding to their respective affinity constants at hPAT1. The basolateral to apical flux of L-[3H]proline was only 8% of that in the opposite direction. Apical uptake of unlabeled L-proline, cis-4-hydroxy-L-proline, and L-azetidine-2-carboxylic acid was stimulated by an inside directed H+ gradient 2- to 3-fold. Total apical to basolateral flux of proline derivatives was moderately correlated with their inhibitory potency for L-[3H]proline uptake and flux inhibition. We conclude that 1) the substrate specificity of hPAT1 is very much broader than so far reported and 2) the system accepts therapeutically relevant proline and GABA derivatives. hPAT1 is a promising candidate for new ways of oral drug delivery.
The findings (a) stereospecific counter transport; (b) equilibrium uptake of L-proline decreased with increasing medium osmolarity; and (c) L-proline and Na+ were taken up into identical intravesicular spaces, indicate that uptake of L-proline by rabbit renal brush border membranes represents transport into membrane vesicles, not surface binding to the membrane. An Na+ gradient between extravesicular and intravesicular media stimulated the initial rate of L-proline uptake about 10 times. Accumulation of the imino acid was maximal at 2 min, then decreased until the equilibrium level was attained. At the peak of this "overshoot" uptake of L-proline was 3-fold greater than the final equilibrium value. These results suggest that the electrochemical Na+ gradient drives the transient movement of L-proline into the membrane vesicles against its concentration gradient. Stimulation of L-proline uptake was specific for Na+. Increasing the Na+ gradient lowered the apparent Km for L-proline. Efflux of L-proline from the membrane vesicles, like uptake, showed stimulatory effects when the Na+ gradient and L-proline were on the same side, and inhibitory effects when the Na+ gradient and the imino acid were on opposite sides of the membrane. Uptake of L-proline, at a given concentration, reflected the sum of contributions from Na+ gradient-dependent and -independent transport systems. The dependent system was saturated at 4 mM L-proline. The independent system did not saturate but may represent the sum of passive diffusion and a "carrier"-mediated system. At physiological concentrations the rate of the Na+ gradient-dependent uptake was 5 times that in the absence of the gradient. In K+-loaded vesicles, valinomycin, but not nigericin, enhanced the Na+ gradient-dependent uptake of L-proline. Gramicidin diminished uptake. These findings indicate that the Na+ gradient-dependent transport of L-proline is an electrogenic process and suggest that the membrane potential is a determinant of L-proline transport. The Na+ gradient-dependent rate of L-proline uptake was strongly inhibited by other imino acids, suggesting that L-imino acids have a common transport system. Glycine and neutral amino acids inhibited the rate of L-proline uptake moderately, acidic amino acid and D-glucose were very weak inhibitors, and basic amino acids were without effect. In the absence of Na+, the rate of L-proline uptake was independent of the presence of other amino acids. These findings indicate that the brush border membrane is a site of amino acid recognition during vectorial transepithelial transport. It is proposed that imino and neutral amino acids inhibit transport of L-proline by competitive interaction with the L-proline "carriers" in addition to competition for the electrochemical Na+ gradient or membrane potential. The findings are relevant to the understanding of genetic amino acid transport disorders, such as iminoglycinuria.
Effects of proline isomerizations on the equilibrium unfolding and kinetic refolding of staphylococcal nuclease were studied by circular dichroism in the peptide region (225 nm) and fluorescence spectra of a tryptophan residue. For this purpose, four single mutants (P11A, P31A, P42A, and P56A) and four multiple mutants (P11A/P47T/P117G, P11A/P31A/P47T/P117G, P11A/P31A/P42A/P47T/P117G, and P11A/P31A/P42A/P47T/P56A/P117G) were constructed. These mutants, together with the single and double mutants for Pro47 and Pro117 constructed in our previous study, cover all six proline sites of the nuclease. The P11A, P31A, and P42A mutations did not change the stability of the protein remarkably, while the P56A mutation increased protein stability to a small extent by 0.5 kcal/mol. The refolding kinetics of the protein were, however, affected remarkably by three of the mutations, namely, P11A, P31A, and P56A. Most notably, the amplitude of the slow phase of the triphasic refolding kinetics of the nuclease observed by stopped-flow circular dichroism decreased by increasing the number of the proline mutations; the slow phase disappeared completely in the proline-free mutant (P11A/P31A/P42A/P47T/P56A/P117G). The kinetic refolding reactions of the wild-type protein assessed in the presence of Escherichia coli cyclophilin A showed that the slow phase was accelerated by cyclophilin, indicating that the slow phase was rate-limited by cis-trans isomerization of the proline residues. Although the fast and middle phases of the refolding kinetics were not affected by cyclophilin, the amplitude of the middle phase decreased when the number of the proline mutations increased; the percent amplitudes for the wild-type protein and the proline-free mutants were 43 and 13%, respectively. In addition to these three phases detected with stopped-flow circular dichroism, a very fast phase of refolding was observed with stopped-flow fluorescence, which had a shorter dead time (3.6 ms) than the stopped-flow circular dichroism. The following conclusions were drawn. (1) The effects of the P11A, P31A, and P56A mutations on the refolding kinetics indicate that the isomerizations of the three proline residues are rate-limiting, suggesting that the structures around these residues (Pro11, Pro31, and Pro56) may be organized at an early stage of refolding. (2) The fast phase corresponds to the refolding of the native proline isomer, and the middle phase whose amplitude has decreased when the number of proline mutations was increased may correspond to the slow refolding of non-native proline isomers. The occurrence of the fast- and slow-refolding reactions together with the slow phase rate-limited by the proline isomerization suggests that there are parallel folding pathways for the native and non-native proline isomers. (3) The middle phase did not completely disappear in the proline-free mutant. This suggests that the slow-folding isomer is produced not only by the proline isomerizations but also by another conformational event that is not related to the prolines. (4) The very fast phase detected with the fluorescent measurements suggests that there is an intermediate at a very early stage of kinetic refolding.
L-Proline can serve as sole source of carbon and nitrogen for the growth of Escherichia coli K12 and other Enterobacteria. L-Proline uptake and L-proline oxidase are subject both to catabolite repression and to specific induction by L-proline or glycyl-L-proline, although their regulation is not strictly coordinate. A strain defective for L-proline uptake due to a lesion at the locus putP does not show elevated uptake activity either on relief of catabolite repression or when grown on glycyl-L-proline as nitrogen source. The apparent Km for L-proline uptake decreases up to 14-fold as uptake Vm increases when cells are induced for both L-proline uptake and L-proline oxidase; cells with increased uptake activity, alone, do not show an altered Km. Although L-proline is metabolized during the uptake measurements, uptake is always active. The observed variations in uptake Km are unlikely to result from the escape of radioactive L-proline metabolites or from reversal of the transport reaction during the uptake measurements. We conclude that the L-proline porter encoded in putP is responsible for 80 to 90% of the constitutive and for the inducible L-proline uptake activity of wild-type bacteria. Although this porter is amplified in cells induced for L-proline catabolism, the observed values for uptake Vm may not be taken as a direct indicator of porter concentration.
Using the method of isomer-specific proteolysis (ISP), the cis-trans nature of the peptide bonds involving prolines-114 and -117 in ribonuclease (RNase) has been investigated. These studies involve the pretreatment of RNase first with either a short pepsin pulse or a short mercaptoethanol pulse to irreversibly unfold the protein and then with a short chymotrypsin pulse to quickly cleave the Tyr115-Val116 bond so that the chain is suitably trimmed for the subsequent stereospecific cleavage either by aminopeptidase P, to investigate proline-117, or by a proline-specific endopeptidase, to investigate proline-114. The most reasonable interpretation of our results suggests that proline-117 is essentially 100% trans in both the native and unfolded states, so it apparently makes no direct contribution to the slow refolding kinetics of RNase. It is also determined that proline-114 is 100% cis in native RNase and ca. 95% cis in reversibly unfolded RNase so only 5% of the unfolded RNase can be rate limited by trans to cis isomerization of proline-114 during refolding. Careful spectroscopic studies of refolding show that the smallest and slowest of the refolding phases, the ct phase, has the proper amplitude (5%), relaxation time (400 s at 10 degrees C), and activation energy (17 kcal) for a phase that is rate limited by the trans to cis isomerization of proline-114. Measurements of the kinetics of binding of cytidine 2'-monophosphate during refolding further show that RNase does not become active until proline-114 has isomerized to the native cis configuration. It is concluded that none of the three prolines thus far examined (i.e., prolines-93, -114, and -117) by the ISP method is involved in the formation of a fully active, nativelike intermediate which has "incorrect" proline isomers. The specific structural process which is responsible for the largest of the three slow refolding phases, the XY phase, is still undetermined. Although ISP results on proline-42 are not yet available, it seems possible that this slow phase may be rate limited by a process other than proline isomerization. In unrelated studies, results from chymotrypsin hydrolyses of several short peptides containing the sequence -X-Y-Pro- show that cleavage of an active X-Y bond is very slow when it is immediately adjacent on the amino side of a proline peptide bond. Thus, chymotrypsin cleavage may not be generally useful as the analytical step in isomer-specific proteolysis.
1. The growth of Escherichia coli proline auxotrophs on medium containing L-proline (50 microgram/ml) induces catabolic enzymes. A bioradiological assay system for proline, using proB cells of E. coli, might give erroneous results owing to proline catabolism by the proline auxotrophs on which the assay depends. 2. Differential utilization of proline and 1-pyrroline-5-carboxylate by the proB cells for the synthesis of protein, and failure of the method to distinguish between these two possible products of the proline-biosynthetic enzymes, might also give rise to error. 3. The proline-dependent incorporation of [14C]phenylalanine into the protein of proline-starved proB auxotrophs was to some degree directly influenced by the presence of crude cell extract from E. coli, even though this was not supplied with substrate and cofactors, and could thus not itself synthesize proline. 4. The kinetics of proline biosynthesis by cell-free extracts were linear and biphasic, only the last phase being affected by the concentrations of substrate and extract. This phenomenon is not understood. 5. Proline biosynthesis is inhibited, not only by high concentrations of ATP, but also by aspartate, glycine, alanine and serine, aspartate having the greatest effect. 6. Attempts at complementation in vitro between extracts of proline auxotrophic mutants were not successful, suggesting the possibility that strain X680 (proA) and/or X278 (proB) may be a double mutant. 7. The enzymes of proline biosyntehsis are co-eluted from a column of Bio-Gel A1.5M in a position corresponding to a mol.wt. of 350000. 8. Comparisons between rates of proline biosynthesis in vivo and in vitro were made.
Excised bean (Phaseolus vulgaris) leaves were used to measure changes in proline content and proline metabolism during rehydration in the dark after the leaves had been incubated in the dark 24 hours in a wilted condition.The increase in nonprotein proline which occurs in wilted leaves stopped immediately upon rehydration, and thereafter levels of proline declined. The rate of decline and the fate of the metabolized proline depended on the amount of carbohydrate present. When the level of carbohydrate was relatively high, the rate of decline in proline content was slow, and the proline was converted mainly to protein proline. When the level of carbohydrate was relatively low, the rate of proline decline was rapid and a large percentage of the proline was oxidized to CO(2) and other amino acids in addition to conversion to protein proline.The results indicate that nonprotein proline can be oxidized when the level is increased by endogenous synthesis. Proline oxidation has been observed when the levels are increased by adding exogenous proline. Oxidation of endogenous proline is inhibited by carbohydrates in the leaf, as is true of oxidation of exogenous proline.
Regulation of A system amino acid transport was studied in primary cultures of the R3230AC mammary adenocarcinoma. Higher rates of carrier-mediated Na+-dependent proline transport, vc, was decreased and was attributed to a two-fold decrease in Vmax and a two-fold increase in Km. When compared to cells grown in standard media (Eagle's minimal essential medium, MEM), cells grown in media supplemented with A system substrates (alanine, serine, glycine, and proline) demonstrated adaptive decreases in proline transport; the decrease was due to two-fold reduction in Vmax, with no change in Km for proline. Even in the presence of preferred substrates for the A system, a density-dependent decrease in proline transport was manifested. Both fast- and slow-growing cultures maintained in MEM exhibited rapid increases in proline transport when switched to buffers devoid of amino acids; two-fold increases in Vmax were seen within 4 hr, but Km was unchanged. This starvation-induced adaptation was completely prevented by inclusion in the buffer of 10 mM proline, 0.1 mM alpha-(methylamino)-isobutyric acid (MetAIB) or 10 mM serine, whereas inclusion of the poorer A system substrate, phenylalanine (10 mM), had no effect. The effects of MetAIB to prevent starvation-induced increases in proline transport were dose-related, rapid, and reversible. Amino acid starvation-induced increases in proline transport were partially blocked by cycloheximide or actinomycin D. Data were obtained demonstrating a temporal relationship between increasing intracellular [proline] and decreasing vc for proline uptake. In addition, efflux of proline from preloaded cells preceded the increase in initial rates of proline entry. Taken together, we concluded that: 1) A system transport in primary cultures of this mammary adenocarcinoma is regulated by cell density as well as by availability of A system substrates, but these two types of regulation are kinetically distinct; and 2) starvation-induced enhancement of proline transport appears to be due to release from transinhibition, but may also involve a derepression-repression type of mechanism.
The isotope effects for the interconversion of L-proline and D-proline, catalyzed by proline racemase, have been determined in the saturated region with both [2-2H]proline and [2-3H]proline. The deuterium fractionation factors for each of the protons in flight have been obtained from two kinds of experiment: by measuring the rate of racemization of one [2-2H]proline enantiomer as it racemizes into an equilibrated pool of unlabeled proline and by measuring the deuterium content of a proline sample at the optical rotation maximum that occurs when an equimolar mixture of one deuterium-labeled enantiomer and the other unlabeled enantiomer runs to equilibrium. The tritium fractionation factors for each of the protons in flight have been determined from measurements of the rate of loss of tritium to the solvent as one [2-3H]proline enantiomer runs to equilibrium. Good agreement is found among the fractionation factors determined by each method. The deuterium fractionation factors for the two protons are not identical: that for the proton derived from L-proline is 0.375 and that for the proton derived from D-proline is 0.44. This difference has been confirmed by a double-competition experiment in which the optical rotation of a mixture of DL-[2-2H]proline and unlabeled DL-proline is followed with time. The rotation (initially zero) passes through a maximum, from which the ratio of the two fractionation factors (0.86) is obtained. These data, coupled with the equilibrium fractionation factor for the 2-position of proline (which has been determined to be 1.17), provide the transition-state factors for each of the in-flight protons, and delineate the nature of the transition state(s) for the enzyme-catalyzed racemization.
The characteristics of L-proline transport in the procyclic form of Trypanosoma brucei were studied by using L-[14C]proline and a quick separation technique by centrifugation through an oil mixture. L-Proline uptake displayed typical Michaelis-Menten kinetics, with a Km of 19 microM and a maximum transport velocity of 17 nmol/min per 10(8) cells at 27 degrees C. The maximum concentration gradient factor obtained after 1 min of incubation was 270-fold in 0.02 mM proline. Cells permeabilized with 80 microM digitonin were still able to accumulate 14C label, but to a lower extent. The temperature-dependence of proline uptake gave an apparent activation energy of 74.9 kJ.mol-1. In competition studies with a 10-fold excess of structural analogues, L-alanine, L-cysteine and L-azetidine-2-carboxylate were found to inhibit L-proline uptake. Variation of pH or addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone ('CCCP') did not affect proline transport, showing that it is not driven by a protonmotive force. The absence of Na+, with or without monensin, did not affect proline transport. The absence of K+ and the addition of the Na+,K(+)-ATPase inhibitor ouabain had no significant effect on proline uptake activity. The thiol-modifying reagent iodoacetate (10 mM) decreased proline uptake by half. KCN (1 mM) inhibited proline uptake to a lesser extent, and the degree of inhibition was proportional to the intracellular ATP concentration. Preliminary experiments on proline transport in plasma-membrane vesicles of the cells, using a filtration technique, showed an uptake of proline (0.67 nmol/mg of protein) by the vesicles, but only in the presence of intravesicular ATP. The results thus obtained suggest that the proline carrier system in T. brucei is ATP-driven and independent of Na+, K+ or H+ co-transport.
The growing zone of maize seedling primary roots accumulates proline at low water potential. Endosperm removal and excision of root tips rapidly decreased the proline pool and greatly reduced proline accumulation in root tips at low water potential. Proline accumulation was not restored by exogenous amino acids. Labelling root lips with [14C]glutamate and [14C]proline showed that the rate of proline utilization (oxidation and protein synthesis) exceeded the rate of biosynthesis by five-fold at high and low water potentials. This explains the reduction in the proline pool following root and endosperm excision and the inability to accumulate proline at low water potential. The endosperm is therefore the source of the proline that accumulates in the root tips of intact seedlings. Proline constituted 10% of the amino acids released from the endosperm. [14C]Proline was transported from the scutellum to other parts of the seedling and reached the highest concentration in the root tip. Less [14C]proline was transported at low water potential but because of the lower rate of protein synthesis and oxidation, more accumulated as proline in the root tip. Despite the low biosynthesis capacity of the roots, the extent of proline accumulation in relation to water potential is precisely controlled by transport and utilization rate.