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The Journal of Biological Chemistry Feb 1999The in vitro folding of Escherichia coli alkaline phosphatase (AP) from the guanidine hydrochloride (GdnHCl) denatured state is characterized by a significant slow phase...
The in vitro folding of Escherichia coli alkaline phosphatase (AP) from the guanidine hydrochloride (GdnHCl) denatured state is characterized by a significant slow phase in the post activational recovery of native protein lability (probed by the susceptibility to GdnHCl denaturation and occurring on the time scale of days) as well as a slow phase in the recovery of activity (on the time scale of minutes). Slow folding events have often been attributed to cis-trans isomerizations of X-Pro peptide bonds, a plausible explanation for AP, which contains 21 prolines per subunit. To investigate the role of proline isomerization in the two measures of refolding mentioned above, we have performed "double-jump" GdnHCl denaturation/renaturation experiments, with a third jump, where the rate of unfolding of refolded protein upon exposure to denaturant was added to assess the rate of change of lability. Our measurements of the time evolution of both the lability and the reactivation of refolded AP as a function of denaturation time show that proline isomerization is unlikely to be the cause of either of these slow events in the refolding of AP. The conclusions are further confirmed by the absence of proline isomerization effects when AP is refolded in the presence of human and periplasmic E. coli peptidyl-prolyl isomerase.
Topics: Alkaline Phosphatase; Enzyme Activation; Humans; Isomerism; Kinetics; Proline; Protein Conformation; Protein Denaturation; Protein Folding; Substrate Specificity
PubMed: 9988686
DOI: 10.1074/jbc.274.8.4532 -
Journal of Chromatography. A Jul 2007Proline derivatives, such as Boc-proline, Boc-2-methylproline, Boc-2-methylproline benzyl ester and Boc-2-methyl-4-hydroxy-proline benzyl ester, have been widely used as...
Proline derivatives, such as Boc-proline, Boc-2-methylproline, Boc-2-methylproline benzyl ester and Boc-2-methyl-4-hydroxy-proline benzyl ester, have been widely used as a building block leading to a variety of pharmaceutical compounds. Therefore, there is a wide interest in the chiral separation of these compounds. High-performance liquid chromatography (HPLC) methods were developed using a Chiralpak AD-H column to separate enantiomers of these proline derivatives. The effect of mobile phase composition and column temperature was studied. For the proline derivatives studied in this work, good resolution was achieved using a mobile phase composition of hexane, ethanol and 0.1% TFA. For prolines containing carboxyl or hydroxy group, resolution was changed dramatically corresponding to changes as little as 1% of ethanol in the mobile phase, suggesting that the dominant chiral recognition is from hydrogen bonding interactions. On the other hand, for prolines containing a benzyl ester instead of hydroxy group next to the chiral center, resolution was not affected as significantly with the changes of ethanol content in the mobile phase, indicating a different leading chiral recognition mechanism, such as inclusion, steric effect, or possible pi-pi interaction. Linearity, precision and limit of detection were also measured for Boc-2-methylproline and Boc-2-methylproline benzyl ester.
Topics: Chromatography, High Pressure Liquid; Polysaccharides; Proline; Stereoisomerism; Temperature
PubMed: 17266975
DOI: 10.1016/j.chroma.2007.01.015 -
Analytical Chemistry Mar 2015The chirality of substituents on an amino acid can significantly change its mode of binding to a metal ion, as shown here experimentally by traveling wave ion mobility...
The chirality of substituents on an amino acid can significantly change its mode of binding to a metal ion, as shown here experimentally by traveling wave ion mobility spectrometry-mass spectrometry (TWIMS-MS) of different proline isomeric molecules complexed with alkali metal ions. Baseline separation of the cis- and trans- forms of both hydroxyproline and fluoroproline was achieved using TWIMS-MS via metal ion cationization (Li(+), Na(+), K(+), and Cs(+)). Density functional theory calculations indicate that differentiation of these diastereomers is a result of the stabilization of differing metal-complexed forms adopted by the diastereomers when cationized by an alkali metal cation, [M + X](+) where X = Li, Na, K, and Cs, versus the topologically similar structures of the protonated molecules, [M + H](+). Metal-cationized trans-proline variants exist in a linear salt-bridge form where the metal ion interacts with a deprotonated carboxylic acid and the proton is displaced onto the nitrogen atom of the pyrrolidine ring. In contrast, metal-cationized cis-proline variants adopt a compact structure where the carbonyl of the carboxylic acid, nitrogen atom, and if available, the hydroxyl and fluorine substituent solvate the metal ion. Experimentally, it was observed that the resolution between alkali metal-cationized cis- and trans-proline variants decreases as the size of the metal ion increases. Density functional theory demonstrates that this is due to the decreasing stability of the compact charge-solvated cis-proline structure with increased metal ion radius, likely a result of steric hindrance and/or weaker binding to the larger metal ion. Furthermore, the unique structures adopted by the alkali metal-cationized cis- and trans-proline variants results in these molecules having significantly different quantum mechanically calculated dipole moments, a factor that can be further exploited to improve the diastereomeric resolution when utilizing a drift gas with a higher polarizability constant.
Topics: Hydroxyproline; Mass Spectrometry; Metals, Alkali; Models, Molecular; Molecular Conformation; Nitrogen; Proline; Protons; Solvents; Stereoisomerism
PubMed: 25664640
DOI: 10.1021/ac5043285 -
Journal of Bacteriology Nov 1998The PheP protein is a high-affinity phenylalanine-specific permease of the bacterium Escherichia coli. A topological model based on genetic analysis involving the...
The PheP protein is a high-affinity phenylalanine-specific permease of the bacterium Escherichia coli. A topological model based on genetic analysis involving the construction of protein fusions with alkaline phosphatase has previously been proposed in which PheP has 12 transmembrane segments with both N and C termini located in the cytoplasm (J. Pi and A. J. Pittard, J. Bacteriol. 178:2650-2655, 1996). Site-directed mutagenesis has been used to investigate the functional importance of each of the 16 proline residues of the PheP protein. Replacement of alanine at only three positions, P54, P341, and P442, resulted in the loss of 50% or more activity. Substitutions at P341 had the most dramatic effects. None of these changes in transport activity were, however, associated with any defect of the mutant protein in inserting into the membrane, as indicated by [35S]methionine labelling and immunoprecipitation using anti-PheP serum. A possible role for each of these three prolines is discussed. Inserting a single alanine residue at different sites within span IX and the loop immediately preceding it also had major effects on transport activity, suggesting an important role for a highly organized structure in this region of the protein.
Topics: Alanine; Amino Acid Sequence; Amino Acid Transport Systems, Neutral; Bacterial Proteins; Cell Membrane; Conserved Sequence; Escherichia coli; Membrane Transport Proteins; Molecular Sequence Data; Mutagenesis, Site-Directed; Proline; Sequence Homology, Amino Acid; Structure-Activity Relationship
PubMed: 9791098
DOI: 10.1128/JB.180.21.5515-5519.1998 -
Journal of the American Chemical Society Feb 2013A recent ion mobility-mass spectrometry (IM-MS) study of the nonapeptide bradykinin (BK, amino acid sequence...
A recent ion mobility-mass spectrometry (IM-MS) study of the nonapeptide bradykinin (BK, amino acid sequence Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)) found evidence for 10 populations of conformations that depend upon the solution composition [J. Am. Chem. Soc. 2011, 133, 13810]. Here, the role of the three proline residues (Pro(2), Pro(3), and Pro(7)) in establishing these conformations is investigated using a series of seven analogue peptides in which combinations of alanine residues are substituted for prolines. IM-MS distributions of the analogue peptides, when compared to the distribution for BK, indicate the multiple structures are associated with different combinations of cis and trans forms of the three proline residues. These data are used to assign the structures to different peptide populations that are observed under various solution conditions. The assignments also show the connectivity between structures when collisional activation is used to convert one state into another.
Topics: Bradykinin; Gases; Isomerism; Molecular Conformation; Proline; Spectrometry, Mass, Electrospray Ionization
PubMed: 23373819
DOI: 10.1021/ja3114505 -
Journal of the American Chemical Society Aug 2006Ion mobility measurements, combined with molecular mechanics simulations, are used to study enantiopure and racemic proline clusters formed by electrospray ionization....
Ion mobility measurements, combined with molecular mechanics simulations, are used to study enantiopure and racemic proline clusters formed by electrospray ionization. Broad distributions of cluster sizes and charge states are observed, ranging from clusters containing only a few proline units to clusters that contain more than 100 proline units (i.e., protonated clusters of the form [xPro + nH](n+) with x = 1 to >100 and n = 1-7). As the sizes of clusters increase, there is direct evidence for nanometer scale, chirally induced organization into specific structures. For n = 4 and 5, enantiopure clusters of approximately 50 to 100 prolines assemble into structures that are more elongated than the most compact structure that is observed from the racemic proline clusters. A molecular analogue, cis-4-hydroxy-proline, displays significantly different behavior, indicating that in addition to the rigidity of the side chain ring, intermolecular interactions are important in the formation of chirally directed clusters. This is the first case in which assemblies of chirally selective elongated structures are observed in this size range of amino acid clusters. Relationships between enantiopurity, cluster shape, and overall energetics are discussed.
Topics: Mass Spectrometry; Models, Molecular; Molecular Conformation; Proline
PubMed: 16910678
DOI: 10.1021/ja0622711 -
Journal of the American Chemical Society Aug 2002The structure of the complex between the heptapeptide Gln-Gly-Arg-Pro-Pro-Gln-Gly and the polyphenol (-)-epigallocatechin gallate (EGCG) has been determined using...
The structure of the complex between the heptapeptide Gln-Gly-Arg-Pro-Pro-Gln-Gly and the polyphenol (-)-epigallocatechin gallate (EGCG) has been determined using time-averaged nuclear Overhauser effects. Effective parameters for the force constant and time constant have been derived, allowing rapid and efficient calculation of structures that satisfy the input restraints. By using multiple start conformations, it is shown that conformational space is covered adequately and that the complex exists in one major conformation, in which the A ring of the EGCG is positioned over Pro5 and the D ring is over Pro4, with the B ring frequently close to the arginine side chain. Alternative conformations are also found, in which the prolines are almost always both involved in stacking interactions, with a strong preference for Pro4 to be involved. The structures are consistent with previous models for the interaction and suggest how precipitation of the complex could occur, which leads to the oral phenomenon of astringency. The method has promise as a general way of docking ligands onto receptors.
Topics: Amino Acid Sequence; Catechin; Nuclear Magnetic Resonance, Biomolecular; Oligopeptides; Proline; Protein Conformation
PubMed: 12175251
DOI: 10.1021/ja0126374 -
European Journal of Dermatology : EJD Apr 2013Wrinkles, one of the characteristics of chronic sun-damaged and/or aged skin, are associated with psychological distress. Apart from the deterioration of collagen and... (Randomized Controlled Trial)
Randomized Controlled Trial
Wrinkles, one of the characteristics of chronic sun-damaged and/or aged skin, are associated with psychological distress. Apart from the deterioration of collagen and elastic fibers in the dermis, which induces the loss of skin elasticity, it has been recently proposed that decreased flexibility or elasticity of the stratum corneum (SC) is also correlated with wrinkle formation. The elasticity of the SC has been shown to be regulated, at least in part, by the amounts and types of amino acids. To evaluate the ability of our newly developed amino acid-derivative (1-carbamimidoyl-L-proline; CLP), which recovers the elastic properties of the SC ex vivo, to improve wrinkles, a clinical test was performed with 126 Japanese female subjects aged 32-50 years who had crow's feet lines on their faces. Three eligible dermatologists evaluated the study according to authorized grades by the Japanese Cosmetic Science Society and scored the subjects who were much improved or improved as 29.7% and 57.8% of all CLP-treated subjects at 4 and 8 weeks, respectively. In contrast, only 1.5% and 8.1% of subjects improved with the placebo lotion at 4 and 8 weeks, respectively. These results suggest a significant efficacy of CLP to improve wrinkles. In parallel with the dermatologists' assessments, skin surface roughness in the CLP-treated group was significantly reduced after treatment with CLP for 4 and 8 weeks compared to the placebo-treated group. The sum of these data suggests that CLP is a promising and useful ingredient for the improvement of wrinkles through its ability to enhance the elasticity of the SC.
Topics: Administration, Topical; Adult; Double-Blind Method; Elasticity; Face; Female; Humans; Middle Aged; Proline; Rheology; Skin; Skin Aging
PubMed: 23568622
DOI: 10.1684/ejd.2013.1964 -
The Journal of Organic Chemistry Jan 2013A selective gold(I)-catalyzed synthesis of chiral aza-proline derivatives has been developed by ring closure of enantioenriched α-hydrazino esters bearing an alkyne...
A selective gold(I)-catalyzed synthesis of chiral aza-proline derivatives has been developed by ring closure of enantioenriched α-hydrazino esters bearing an alkyne group. These are easily prepared through a synthetic strategy involving two key steps: organocatalyzed electrophilic amination of pent-4-ynal with dialkyl azodicarboxylate promoted by l-proline and functionalization of the triple bond by Sonogashira cross-coupling. This strategy allowed the preparation of a range of enantioenriched α-hydrazino esters that underwent ring closure by using Ph(3)PAuCl/AgBF(4) as a catalytic system. Under these conditions, 5-exo-dig cyclization was favored over 6-endo-dig and aza-proline derivatives were obtained in good yields without epimerization at the stereogenic center. Influence of the catalytic system, hydrazine protecting group and alkyne substitution on the cyclization step has also been investigated.
Topics: Alkynes; Aza Compounds; Catalysis; Cyclization; Esters; Gold; Hydrazines; Molecular Structure; Proline; Stereoisomerism
PubMed: 23167735
DOI: 10.1021/jo302320v -
Analytica Chimica Acta May 2010A new molecularly imprinted polymer (MIP)-chemiluminescence (CL) method has been developed for detection of proline. The molecularly imprinted polymer microspheres were...
A new molecularly imprinted polymer (MIP)-chemiluminescence (CL) method has been developed for detection of proline. The molecularly imprinted polymer microspheres were synthesized using precipitation polymerization with hydroxyproline, a structural analogue, as the template. Polymer microspheres were immobilized in microtiter plates (96 wells) which selectively adsorbed the analyte (dansyl-proline). After washing, the bound fraction was quantified based on peroxyoxalate chemiluminescence (PO-CL) reaction enhanced by imidazole. The cavity of MIP synthesized with hydroxyproline as template is smaller, which can avoid non-specific adsorption and lead to enhancement of specificity, response speed and sensitivity when recognizing dansyl-proline. Under the optimum conditions, the relative CL intensity has a linear relationship with the concentration of proline in the range of 1x10(-6) mol L(-1) to 4x10(-5) mol L(-1), with a limit of detection 3x10(-7) mol L(-1) (3sigma). The relative standard deviation (R.S.D.) for proline (1x10(-6) mol L(-1), n=7) was 3.7%. The MIP-CL method can become a useful analytical technology for determination of proline in real sample.
Topics: Adsorption; Chemical Precipitation; Luminescent Measurements; Microspheres; Molecular Imprinting; Oxalates; Polymers; Proline
PubMed: 20433967
DOI: 10.1016/j.aca.2010.03.031