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Rudolf K Allemann

Publications and source records attributed to Rudolf K Allemann.

At least 19 recordsLinked to original sources
PubMed2006

Protein motions during catalysis by dihydrofolate reductases.

Dihydrofolate reductase (DHFR) maintains the intracellular pool of tetrahydrofolate through catalysis of hydrogen transfer from reduced nicotinamide adenine dinucleotide to 7,8-dihydrofolate. We report results for pre-steady-state kinetic studies of the temperature dependence of the rates and the hydrogen/deuterium-kinetic isotope effects for the reactions catalysed by the enzymes from the mesophilic Escherichia coli and the hyperthermophilic Thermatoga maritima. We propose an evolutionary pattern in which catalysis progressed from a relatively rigid active site structure in the ancient thermophilic DHFR to a more flexible and kinetically more efficient structure in E. coli that actively promotes hydrogen transfer at physiological pH by modulating the tunnelling distance. The E. coli enzyme appeared relatively robust, in that kinetically severely compromised mutants still actively propagated the reaction. The reduced hydrogen transfer rates of the extensively studied Gly121Val mutant of DHFR from E. coli were most likely due to sterically unfavourable long-range effects from the introduction of the bulky isopropyl group.

Bacterial Proteins↗
PubMed2006

Designed high affinity Cu2+-binding alpha-helical foldamer.

The design, synthesis, and characterization of a folded high-affinity metal-binding peptide is described. Based on the previously described folded peptide NTH-18, in which an alpha-helix was constrained through two disulfide bonds to a C-terminal extension of noncanonical secondary structure, a peptide (1) was designed to contain two histidine residues in positions 3 and 7. Air oxidation of 1 led to the formation of peptide 2, which contained two intramolecular disulfide bonds. The presence of the two histidines significantly destabilized the alpha-helical structure of 2 when compared to NTH-18. However, CD spectroscopy revealed that the addition of certain transition metal ions allowed the reformation of a stable alpha-helix. CD, NMR, and EPR spectroscopy as well as MALDI-TOF mass spectrometry indicated that 2 bound to Cu2+ to form a 1:1 complex via the imidazoles of the two histidine side chains. A glycine displacement assay revealed a dissociation constant for this complex of 5 nM at pH 8, which is the lowest reported value for a designed Cu2+-binding peptide. This peptide displayed more than 100-fold selectivity for Cu2+ over Zn2+, Ni2+, and Co2+. The 1.05 A crystal structure of the Cu(II)-complex of 2 revealed a square-pyramidal coordination geometry and confirmed that 2 bound to copper in an alpha-helical conformation via its two histidine side chains. The high affinity metal binding of peptide 2 demonstrates that metals can be used for the selective nucleation of alpha-helices.

Copper↗
PubMed2006

Hydride transfer reaction catalyzed by hyperthermophilic dihydrofolate reductase is dominated by quantum mechanical tunneling and is promoted by both inter- and intramonomeric correlated motions.

Simulations of hydride and deuteride transfer catalyzed by dihydrofolate reductase from the hyperthermophile Thermotoga maritima (TmDHFR) are presented. TmDHFR was modeled with its active homodimeric quaternary structure, where each monomer has three subdomains. The potential energy function was a combined quantum mechanical and molecular mechanical potential (69 atoms were treated quantum mechanically, and 35 287, by molecular mechanics). The calculations of the rate constants by ensemble-averaged variational transition state theory with multidimensional tunneling predicted that hydride and deuteride transfer at 278 K proceeded with 81 and 80% by tunneling. These percentages decreased to 50 and 49% at 338 K. The kinetic isotope effect was dominated by contributions of bound vibrations and decreased from 3.0 to 2.2 over the temperature range. The calculated rates for hydride and deuteride transfer catalyzed by the hypothetical monomer were smaller by approximately 2 orders of magnitude. At 298 K tunneling contributed 73 and 66% to hydride and deuteride transfer in the monomer. The decreased catalytic efficiency of the monomer was therefore not the result of a decrease of the tunneling contribution but an increase in the quasi-classical activation free energy. The catalytic effect was associated in the dimer with correlated motions between domains as well as within and between subunits. The intrasubunit correlated motions were decreased in the monomer when compared to both native dimeric TmDHFR and monomeric E. coli enzyme. TmDHFR and its E. coli homologue involve similar patterns of correlated interactions that affect the free energy barrier of hydride transfer despite only 27% sequence identity and different quaternary structures.

Algorithms↗
PubMed2006

Stabilisation of transition states prior to and following eudesmane cation in aristolochene synthase.

The mechanistic details of the cyclisation of farnesylpyrophosphate (FPP) by aristolochene synthase (AS) from Penicillium roqueforti have only recently begun to emerge, mainly through the analysis of the reaction products generated by AS-mutants. The reaction proceeds through several intermediates including germacrene A and eudesmane cation. Previous work suggested that the side chain of phenylalanine 178 promoted the conversion of eudesmane cation to aristolochene. We now report that the catalytic function of this residue during the conversion of eudesmane cation to aristolochene is mainly due to the large size of its side chain, which facilitates the hydride shift from C2 to C3, rather than its aromatic character. In addition, F178 appears to control the regioselectivity of the final deprotonation step and, together with F112, helps stabilise the developing positive charge on C1 after the expulsion of pyrophosphate from the substrate. These results complete a screen of likely active-site aromatic residues and establish their respective roles in the conversion of FPP to aristolochene.

Base Sequence↗
PubMed2006

Coupling of protein motions and hydrogen transfer during catalysis by Escherichia coli dihydrofolate reductase.

The enzyme DHFR (dihydrofolate reductase) catalyses hydride transfer from NADPH to, and protonation of, dihydrofolate. The physical basis of the hydride transfer step catalysed by DHFR from Escherichia coli has been studied through the measurement of the temperature dependence of the reaction rates and the kinetic isotope effects. Single turnover experiments at pH 7.0 revealed a strong dependence of the reaction rates on temperature. The observed relatively large difference in the activation energies for hydrogen and deuterium transfer led to a temperature dependence of the primary kinetic isotope effects from 3.0+/-0.2 at 5 degrees C to 2.2+/-0.2 at 40 degrees C and an inverse ratio of the pre-exponential factors of 0.108+/-0.04. These results are consistent with theoretical models for hydrogen transfer that include contributions from quantum mechanical tunnelling coupled with protein motions that actively modulate the tunnelling distance. Previous work had suggested a coupling of a remote residue,Gly121, with the kinetic events at the active site. However, pre-steady-state experiments at pH 7.0 with the mutant G121V-DHFR, in which Gly121 was replaced with valine, revealed that the chemical mechanism of DHFR catalysis was robust to this replacement. The reduced catalytic efficiency of G121V-DHFR was mainly a consequence of the significantly reduced pre-exponential factors, indicating the requirement for significant molecular reorganization during G121V-DHFR catalysis. In contrast, steady-state measurements at pH 9.5, where hydride transfer is rate limiting, revealed temperature-independent kinetic isotope effects between 15 and 35 degrees C and a ratio of the pre-exponential factors above the semi-classical limit, suggesting a rigid active site configuration from which hydrogen tunnelling occurs. The mechanism by which hydrogen tunnelling in DHFR is coupled with the environment appears therefore to be sensitive to pH.

Catalysis↗
PubMed2005

De novo design of a stable N-terminal helical foldamer.

A peptide NTH-18 was synthesized in which a N-terminal helix is stabilised by two crossed disulfide bonds to a C-terminal extension. The design was inspired by the structure of the neurotoxic peptide apamin, which has previously been used to stabilise helices in miniature enzymes. CD- and NMR-spectroscopy indicated that NTH-18 adopted a fold similar to that found in apamin. However, the arrangement of the elements of secondary structures was inverted relative to apamin; a N-terminal alpha-helix was connected by a reverse turn to a C-terminal extension of non-canonical secondary structure. NTH-18 displayed significant stability to heat and changes of pH. The high definition of the N-terminal end of the alpha-helix of NTH-18 should make this peptide a useful vehicle to stabilise alpha-helices in proteins with applications in protein engineering and molecular recognition.

Amino Acid Sequence↗
PubMed2005

Photocontrol of DNA binding specificity of a miniature engrailed homeodomain.

Control of DNA binding of HDH-3, a 18-residue polypeptide based on the recognition helix of the Q50K engrailed homeodomain, has been achieved. HDH-3 was linked to an azobenzene cross-linker through two cysteine residues in an i, i + 11 spacing. For the thermodynamically stable trans configuration of the cross-linker, the dark-adapted peptide (dad-HDH-3) adopted a mainly alpha-helical structure as judged by circular dichroism (CD) spectroscopy. After irradiation with light of 360 nm, the helical content of the peptide (irrad-HDH-3) was reduced significantly and the CD spectrum of the irradiated peptide resembled that of the largely unstructured, unalkylated peptide. Despite lacking helices-1 and -2 and the N-terminal arm of Q50K engrailed, dad-HDH-3 bound to its natural DNA target sequence TAATCC (QRE) with high affinity (K(D) = 7.5 +/- 1.3 nM). The binding affinity for the mutant DNA sequence, TAATTA (ERE), was reduced significantly (K(D) = 140 +/- 11 nM). Unlike irrad-HDH-3, which like the unalkylated parent peptide displayed only marginal DNA binding specificity, dad-HDH-3 specified base pairs 5 and 6 of QRE with an accuracy rivaling that of the intact wild-type Q50K engrailed homeodomain, making dad-HDH-3 the most specific designed DNA binding miniature homeodomain reported to date. Moreover, DNA binding affinity and specificity of HDH-3 could be controlled externally by irradiation with light.

Azo Compounds↗
PubMed2004

Synthesis of homogenous site-selectively glycosylated proteins.

Apparently homogenous glycoproteins can be synthesised in good yield by a combination of site directed mutagenesis, a highly flexible but selective chemical derivatisation and efficient purification through the use of glycosyl thiosulfonates such as 2-((biotinoyl)-amino)-ethyl methanethiosulfonate.

Carbohydrates↗
PubMed2004

A stable miniature protein with oxaloacetate decarboxylase activity.

An 18-residue miniature enzyme, Apoxaldie-1, has been designed, based on the known structure of the neurotoxic peptide apamin. Three lysine residues were introduced on the solvent-exposed face of the apamin alpha-helix to serve as an active site for decarboxylation of oxaloacetate. The oxidised form of Apoxaldie-1, in which two disulfide bonds stabilise the alpha-helix, formed spontaneously. CD spectroscopy measurements revealed that, in its oxidised form, Apoxaldie-1 adopted a stably folded structure, which was lost upon reduction of the disulfide bonds. Despite its small size and the absence of a designed binding pocket, Apoxaldie-1 displayed saturation kinetics in its oxidised form and catalysed the decarboxylation of oxaloacetate at a rate that was almost four orders of magnitude faster than that observed with n-butylamine. This rivals the performance of the best synthetic oxaloacetate decarboxylases reported to date. Unlike those, however, Apoxaldie-1 displayed significant stability. It maintained its secondary structure at temperatures in excess of 75 degrees C, in the presence of high concentrations of guanidinium chloride and at pH values as low as 2.2. Apamin-based catalysts have potential for the generation of miniature peptides that display activity under nonphysiological conditions.

Amino Acid Sequence↗
PubMed2004

Dual role for phenylalanine 178 during catalysis by aristolochene synthase.

A mutant of aristolochene synthase, in which Phe 178 was replaced by Val, produced significant amounts of alpha-and beta-farnesene as well as alpha and beta-selinene and selina-4,11-diene, suggesting that Phe 178 is involved in the stabilisation of transition states preceding germacrene A and following eudesmane cation.

Amino Acid Substitution↗
PubMed2004

Nucleophilic and general acid catalysis at physiological pH by a designed miniature esterase.

A 31-residue peptide (Art-Est) was designed to catalyse the hydrolysis of p-nitrophenyl esters through histidine catalysis on the solvent exposed face of the alpha-helix of bovine pancreatic polypeptide. NMR spectroscopy indicated that Art-Est adopted a stable 3-dimensional structure in solution. Art-Est was an efficient catalyst with second order rate constants of up to 0.050 M(-1) s(-1). The activity of Art-Est was a consequence of the increased nucleophilicity of His-22, which had a reduced pK(a) value of 5.5 as a consequence of its interaction with His-18 and the positively charged Arg-25 and Arg-26. Mass spectrometry and NMR spectroscopy confirmed that the Art-Est catalysed hydrolysis of p-nitrophenyl esters proceeded through an acyl-enzyme intermediate. A solvent kinetic isotope effect of 1.8 indicated that the transition state preceding the acyl intermediate was stabilised through interaction with the protonated side-chain of His-18 and indicated a reaction mechanism similar to that generally observed for natural esterases. The involvement in the reaction of two histidine residues with different pK(a) values led to a bell-shaped dependence of the reaction rate on the pH of the solution. The catalytic behaviour of Art-Est indicated that designed miniature enzymes can act in a transparent mechanism based fashion with enzyme-like behaviour through the interplay of several amino acid residues.

Amino Acid Sequence↗
PubMed2004

Pivotal role of Gly 121 in dihydrofolate reductase from Escherichia coli: the altered structure of a mutant enzyme may form the basis of its diminished catalytic performance.

The structure and folding of dihydrofolate reductase (DHFR) from Escherichia coli and the mutant G121V-DHFR, in which glycine 121 in the exterior FG loop was replaced with valine, were studied by molecular dynamics simulations and CD and fluorescence spectroscopy. The importance of residue 121 for the chemical step during DHFR catalysis had been demonstrated previously. High-temperature MD simulations indicated that while DHFR and G121V-DHFR followed similar unfolding pathways, the strong contacts between the M20 loop and the FG loop in DHFR were less stable in the mutant. These contacts have been proposed to be involved in a coupled network of interactions that influence the protein dynamics and promote catalysis [Benkovic, S. J., and Hammes-Schiffer, S. (2003) Science 301, 1196-1202]. CD spectroscopy of DHFR and G121V-DHFR indicated that the two proteins existed in different conformations at room temperature. While the thermally induced unfolding of DHFR was highly cooperative with a midpoint at 51.6 +/- 0.7 degrees C, G121V-DHFR exhibited a gradual decrease in its level of secondary structure without a clear melting temperature. Temperature-induced unfolding and renaturation from the urea-denatured state revealed that both proteins folded via highly fluorescent intermediates. The formation of these intermediates occurred with relaxation times of 149 +/- 4.5 and 256 +/- 13 ms for DHFR and G121V-DHFR, respectively. The fluorescence intensity for the intermediates formed during refolding of G121V-DHFR was approximately twice that of the wild-type. While the fluorescence intensity then slowly decayed for DHFR toward a state representing the native protein, G121V-DHFR appeared to be trapped in a highly fluorescent state. These results suggest that the reduced catalytic activity of G121V-DHFR is the consequence of nonlocal structural effects that may result in a perturbation of the network of promoting motions.

Catalysis↗
PubMed2004

Removal of the phosphate group in mechanism-based inhibitors of inositol monophosphatase leads to unusual inhibitory activity.

Inositol monophosphatase is widely held to be the therapeutic target for inhibition by lithium ion in the treatment of bipolar disorder. In a continued effort to improve the bioavailability of alternative inhibitors, we have designed and tested two new series of compounds; phosphonates and product-like mimics. Phosphonate substrate mimics were competitive inhibitors of reduced potency as compared to phosphate based inhibitors. Product mimics however, showed various inhibitory modes of action. The 6-butylamino derivative 6p was an uncompetitive inhibitor when acting alone (K(i)= 0.3 mM) but displayed non-competitive inhibition in the presence of inorganic phosphate. This compound represents a new lead in the search for a viable replacement for lithium ion therapy.

Animals↗
PubMed2004

Controlling the DNA binding specificity of bHLH proteins through intramolecular interactions.

Reversible control of the conformation of proteins was employed to probe the relationship between flexibility and specificity of the basic helix-loop-helix protein MyoD. A fusion protein (apaMyoD) was designed where the basic DNA binding helix of MyoD was stablized by an amino-terminal extension with a sequence derived from the bee venom peptide apamin. The disulfide-stabilized helix from apamin served as a nucleus for a helix that extended for a further ten residues, thereby holding apaMyoD's DNA recognition helix in a predominantly alpha-helical conformation. The thermal stability of the DNA complexes of apaMyoD was increased by 13 degrees C relative to MyoD-bHLH. Measurements of the fluorescence anisotropy change on DNA binding indicated that apaMyoD bound to E-box-containing DNA sequences with enhanced affinity relative to MyoD-bHLH. Consequently, the DNA binding specificity of apaMyoD was increased 10-fold.

Amino Acid Sequence↗
PubMed2003

Evidence for environmentally coupled hydrogen tunneling during dihydrofolate reductase catalysis.

Hydride transfer during catalysis by dihydrofolate reductase from Thermotoga maritima has been studied by stopped flow spectroscopy. The reduction of dihydrofolate by NADPH showed a biphasic temperature dependence of the deuterium kinetic isotope effect. At temperatures above 25 degrees C the KIE was temperature independent, while the reaction rates were strongly temperature dependent. Below 25 degrees C the KIE becomes dependent on temperature, and the ratio of the preexponential factors is inverse, suggesting a greater role for active dynamics that modulate the tunneling distance.

Catalysis↗