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The fundamental mechanism of biochemistry lies on the reaction kinetics, which is determined by the reaction pathways. Interestingly, the reaction pathway is a challenging concept for undergraduate students. Experimentally, it is difficult to observe, and theoretically, it requires some degree of physics knowledge, namely statistical and quantum mechanics. However, students can utilize computational methods to study the reaction kinetics without paying too much attention but not wholly neglecting the comprehension of physics. We hereby provided an approach to study the reaction kinetics based on density-functional calculations. We particularized the study of the isomerization case involving five molecules at three different temperatures and emphasized the importance of the transition state in the study of reaction kinetics. The results we presented were in good agreement with the experiments and provided useful insights to assist students in the application of their knowledge into their research., (© 2020 International Union of Biochemistry and Molecular Biology.)

The Hill equation, which models the cooperative ligand-receptor binding equilibrium, turns out to be useful in modeling the progression of infectious disease outbreaks such as CoViD-19. The equation fits well the data for total and daily case numbers, allows tentative predictions for the half-point and end point of the epidemic, and presents a mathematical characterization of how social interventions "flatten the curve" of the disease progression., (© 2020 International Union of Biochemistry and Molecular Biology.)

Four different extraction methods, including hot water extraction (HWE), ultrasonic-assisted extraction (UAE), enzyme-assisted extraction (EAE) and ultrasonic-enzyme-assisted extraction (UEAE), were applied to extract polysaccharides from Trifolium repens L. (TRPs). In addition, response surface methodology (RSM) was performed to optimize the extraction conditions of TRPs. The results showed that different extraction methods had significant effects on the extraction yields and antioxidant activities of TRPs. TRPs extracted by the EAE method (10.57%) and UEAE method (10.62%) had significantly higher extraction yields than TRPs extracted by the HWE method (8.35%) and UAE method (9.43%) (P < 0.05). However, there was no significant difference between the extraction yields of the EAE method and UEAE method (P > 0.05). TRPs extracted by the EAE method had a higher content of uronic acid and exhibited better antioxidant capacities. Therefore, EAE was selected as the optimal extraction method to extract TRPs. The optimal extraction conditions of EAE to extract TPRs were liquid-solid ratio 30 mL/g, enzymolysis time 87 min, enzyme-complex dosage 1.6% and pH 6, leading to a TRPs yield of 13.15%.

The ene reaction involves the addition of an 'ene' to an 'enophile.' The retro-ene reaction is the reverse of the ene reaction. In recent years various biological molecules have been found to form covalent intermediates (ene-adducts) that might be the result of an ene reactions. Such adducts have been characterized or implicated for dihydropyridines and pyridininum cofactors derived from vitamin B3, such as the reduced and oxidized forms of nicotinamide adenine dinucleotide (NADH/NAD); flavin cofactors derived from vitamin B2, such as flavin adenine dinucleotide, FAD, and flavin mononucleotide, FMN; vitamin C; the oxime intermediate of nitric oxide synthase; tyrosine; and other biomolecules. Given the ubiquitous nature of these cofactors, it might be speculated that the formation of ene-adducts is a more common principle in biochemistry., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Metabolism involves numerous reactions and organic compounds that the student must master to understand adequately the processes involved. Part of biochemical learning should include some knowledge of the structure of biomolecules, although the acquisition of such knowledge can be time-consuming and may require significant effort from the student. In this report, we describe the "polygonal model" as a new means of graphically representing biomolecules. This model is based on the use of geometric figures such as open triangles, squares, and circles to represent hydroxyl, carbonyl, and carboxyl groups, respectively. The usefulness of the polygonal model was assessed by undergraduate students in a classroom activity that consisted of "transforming" molecules from Fischer models to polygonal models and vice and versa. The survey was applied to 135 undergraduate Biology and Nursing students. Students found the model easy to use and we noted that it allowed identification of students' misconceptions in basic concepts of organic chemistry, such as in stereochemistry and organic groups that could then be corrected. The students considered the polygonal model easier and faster for representing molecules than Fischer representations, without loss of information. These findings indicate that the polygonal model can facilitate the teaching of metabolism when the structures of biomolecules are discussed. Overall, the polygonal model promoted contact with chemical structures, e.g. through drawing activities, and encouraged student-student dialog, thereby facilitating biochemical learning. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):66-75, 2018., (© 2017 The International Union of Biochemistry and Molecular Biology.)

In 1977, John G. Topliss introduced the Topliss Batchwise Scheme, a straightforward nonmathematical procedure to assist medicinal chemists in optimizing the substitution pattern of a phenyl ring. Despite its long period of application, a thorough validation of this method has been missing so far. Here, we address this issue by gathering 129 congeneric series from the ChEMBL database, suitable to retrospectively assess the approach. Frequency analysis of Topliss' schemes showed that the π, Es, σ, and -σ scheme occurred in 17, 20, 6, and 4 congeneric series, respectively. We observed a significant difference of π scheme frequency in enzymes versus membrane receptors, with 12 versus only 2 occurrences. Validation of Topliss schemes in potency optimization showed a remarkable performance increase after restricting the data set to analogue series tested solely against enzymes. In this setting, the Es and the π scheme were successful in 50% and 56% of the analogue series, respectively.

We have developed a protein loop structure prediction method by combining a new energy function, which we call E PLM (energy for protein loop modeling), with the conformational space annealing (CSA) global optimization algorithm. The energy function includes stereochemistry, dynamic fragment assembly, distance-scaled finite ideal gas reference (DFIRE), and generalized orientation- and distance-dependent terms. For the conformational search of loop structures, we used the CSA algorithm, which has been quite successful in dealing with various hard global optimization problems. We assessed the performance of E PLM with two widely used loop-decoy sets, Jacobson and RAPPER, and compared the results against the DFIRE potential. The accuracy of model selection from a pool of loop decoys as well as de novo loop modeling starting from randomly generated structures was examined separately. For the selection of a nativelike structure from a decoy set, E PLM was more accurate than DFIRE in the case of the Jacobson set and had similar accuracy in the case of the RAPPER set. In terms of sampling more nativelike loop structures, E PLM outperformed E DFIRE for both decoy sets. This new approach equipped with E PLM and CSA can serve as the state-of-the-art de novo loop modeling method.

Enzymes play important roles in catalysing biochemical transaction paths, acting as logical machines through the morphology of the processes. A key challenge in elucidating the nature of these systems, and for engineering manufacturing methods inspired by biochemical reactions, is to attain a comprehensive understanding of the stereochemical ground rules of enzymatic reactions. Here, we present a model of catalysis that can be performed magnetically by centimetre-sized passive floating units. The designed system, which is equipped with permanent magnets only, passively obeys the local causalities imposed by magnetic interactions, albeit it shows a spatial behaviour and an energy profile analogous to those of biochemical enzymes. In this process, the enzyme units trigger physical conformation changes of the target by levelling out the magnetic potential barrier (activation potential) to a funnel type and, thus, induce cascading conformation changes of the targeted substrate units reacting in parallel. The inhibitor units, conversely, suppress such changes by increasing the potential. Because the model is purely mechanical and established on a physics basis in the absence of turbulence, each performance can be explained by the morphology of the unit, extending the definition of catalysis to systems of alternative scales.

The time-evolution of continuous-time discrete-state biochemical processes is governed by the Chemical Master Equation (CME), which describes the probability of the molecular counts of each chemical species. As the corresponding number of discrete states is, for most processes, large, a direct numerical simulation of the CME is in general infeasible. In this paper we introduce the method of conditional moments (MCM), a novel approximation method for the solution of the CME. The MCM employs a discrete stochastic description for low-copy number species and a moment-based description for medium/high-copy number species. The moments of the medium/high-copy number species are conditioned on the state of the low abundance species, which allows us to capture complex correlation structures arising, e.g., for multi-attractor and oscillatory systems. We prove that the MCM provides a generalization of previous approximations of the CME based on hybrid modeling and moment-based methods. Furthermore, it improves upon these existing methods, as we illustrate using a model for the dynamics of stochastic single-gene expression. This application example shows that due to the more general structure, the MCM allows for the approximation of multi-modal distributions.

We investigate numerically pseudo-first-order irreversible bimolecular reactions of the type A + B → B between hard spheres undergoing event-driven Brownian dynamics. We study the encounter rate and the survival probability of A particles as functions of the packing fraction ϕ in the trapping (a single particle diffusing among static non-overlapping traps) and target (many traps diffusing in the presence of a single static target particle) settings, as well as in the case of diffusing traps and particles (full mobility). We show that, since inertial effects are accounted for in our simulation protocol, the standard Smoluchowski theory of coagulation of non-interacting colloids is recovered only at times greater than a characteristic time Δt, marking the transition from the under-damped to the over-damped regime. We show that the survival probability S(t) decays exponentially during this first stage, with a rate 1/τ0 is proportional to φ. Furthermore, we work out a simple analytical expression that is able to capture to an excellent extent the numerical results for t < Δt at low and intermediate densities. Moreover, we demonstrate that the time constant of the asymptotic exponential decay of S(t) for diffusing traps and particles is k(S)(-1), where kS = 4π(DA + DB)Rρ is the Smoluchowski rate. Detailed analyses of the effective decay exponent β = d [log(-logS(t))]/d (logt) and of the steady-state encounter rate reveal that the full mobility and trapping problem are characterized by very similar kinetics, rather different from the target problem. Our results do not allow one to ascertain whether the prediction S(t) is proportional to exp(-at(3/2)) (a = const.) as t → ∞ for the trapping problem in 3D is indeed recovered. In fact, at high density, S(t) is dominated by short encounter times, which makes it exceedingly hard to record the events corresponding to the exploration of large, trap-free regions. As a consequence, at high densities the steady-state rate simply tends to 1/τ0. Finally, we work out an analytical formula for the rate that shows a remarkable agreement with the numerics up φ = 0.4.

Computational enzymology is a rapidly maturing field that is increasingly integral to understanding mechanisms of enzyme-catalyzed reactions and their practical applications. Combined quantum mechanics/molecular mechanics (QM/MM) methods are important in this field. By treating the reacting species with a quantum mechanical method (i.e., a method that calculates the electronic structure of the active site) and including the enzyme environment with simpler molecular mechanical methods, enzyme reactions can be modeled. Here, we review QM/MM methods and their application to enzyme-catalyzed reactions to investigate fundamental and practical problems in enzymology. A range of QM/MM methods is available, from cheaper and more approximate methods, which can be used for molecular dynamics simulations, to highly accurate electronic structure methods. We discuss how modeling of reactions using such methods can provide detailed insight into enzyme mechanisms and illustrate this by reviewing some recent applications. We outline some practical considerations for such simulations. Further, we highlight applications that show how QM/MM methods can contribute to the practical development and application of enzymology, e.g., in the interpretation and prediction of the effects of mutagenesis and in drug and catalyst design.

Site-specific recombination plays key roles in microbe biology and is exploited extensively to manipulate the genomes of higher organisms. Cre is a well studied site-specific recombinase, responsible for establishment and maintenance of the P1 bacteriophage genome in bacteria. During recombination, Cre forms a synaptic complex between two 34-bp DNA sequences called loxP after which a pair of strand exchanges forms a Holliday junction (HJ) intermediate; HJ isomerization then allows a second pair of strand exchanges and thus formation of the final recombinant product. Despite extensive work on the Cre-loxP system, many of its mechanisms have remained unclear, mainly due to the transient nature of complexes formed and the ensemble averaging inherent to most biochemical work. Here, we address these limitations by introducing tethered fluorophore motion (TFM), a method that monitors large-scale DNA motions through reports of the diffusional freedom of a single fluorophore. We combine TFM with Förster resonance energy transfer (FRET) and simultaneously observe both large- and small-scale conformational changes within single DNA molecules. Using TFM-FRET, we observed individual recombination reactions in real time and analyzed their kinetics. Recombination was initiated predominantly by exchange of the "bottom-strands" of the DNA substrate. In productive complexes we used FRET distributions to infer rapid isomerization of the HJ intermediates and that a rate-limiting step occurs after this isomerization. We also observed two nonproductive synaptic complexes, one of which was structurally distinct from conformations in crystals. After recombination, the product synaptic complex was extremely stable and refractory to subsequent rounds of recombination.

We present an exact expression for the mean exit time through the cap of a confining sphere for particles alternating phases of surface and of bulk diffusion. The present approach is based on an integral equation which can be solved analytically. In contrast to the statement of Berezhkovskii and Barzykin [J. Chem. Phys. 136, 54115 (2012)], we show that the mean exit time can be optimized with respect to the desorption rate, under analytically determined criteria.

The linear noise approximation (LNA) offers a simple means by which one can study intrinsic noise in monostable biochemical networks. Using simple physical arguments, we have recently introduced the slow-scale LNA (ssLNA), which is a reduced version of the LNA under conditions of timescale separation. In this paper we present the first rigorous derivation of the ssLNA using the projection operator technique and show that the ssLNA follows uniquely from the standard LNA under the same conditions of timescale separation as those required for the deterministic quasi-steady-state approximation. We also show that the large molecule number limit of several common stochastic model reduction techniques under timescale separation conditions constitutes a special case of the ssLNA.

This review describes the historical difficulties in devising a kinetically satisfactory mechanism for the classical catalase after its identification as a unique catalytic entity in 1902 and prior to the breakthrough 1947 analysis by Chance and co-workers which led to the identification of peroxide compounds I and II. The role of protons in the formation of these two ferryl complexes is discussed and current problems of inhibitory ligand and hydrogen donor binding at the active site are outlined, especially the multiple roles involving formate or formic acid. A previous mechanism of NADPH-dependent catalase protection against substrate inhibition is defended. A revised model linking the catalytic ('catalatic') action and the one-electron side reactions involving compound II is suggested. And it is concluded that, contrary to an idea proposed in 1963 that eukaryotic catalase might be a 'fossil enzyme', current thinking gives it a central role in the redox protective processes of long term importance for human and other eukaryotic and prokaryotic life., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Single molecule force spectroscopy probes the strength, lifetime, and energetic details of intermolecular interactions in a simple experiment. A growing number of these studies have reported distinctly nonlinear trends in rupture force with loading rate that are typically explained in conventional models by invoking complex escape pathways. Recent analyses suggested that these trends should be expected even for simple barriers based on the basic assumptions of bond rupture dynamics and thus may represent the norm rather than the exception. Here we explore how these nonlinear trends reflect the two fundamental regimes of bond rupture: (i) a near-equilibrium regime, produced either by bond reforming in the case of a single bond or by asynchronized rupture of multiple individual bonds, and (ii) a kinetic regime produced by fast, non-equilibrium bond rupture. We analyze both single- and multi-bonded cases, describe the full evolution of the system as it transitions between near- and far-from-equilibrium loading regimes, and show that both interpretations produce essentially identical force spectra. Data from 10 different molecular systems show that this model provides a comprehensive description of force spectra for a diverse suite of bonds over experimentally relevant loading rates, removes the inconsistencies of previous interpretations of transition state distances, and gives ready access to both kinetic and thermodynamic information about the interaction. These results imply that single-molecule binding free energies for a vast number of bonds have already been measured.

Despite the biological relevance of glycosyltrasferases (GTs) and the many efforts devoted to this subject, the catalytic mechanism through which a subclass of this large family of enzymes, namely those that operate with net retention of the anomeric configuration, has not been fully established. Here, we show that in the absence of an acceptor, an archetypal retaining GT such as Pyrococcus abyssi glycogen synthase (PaGS) reacts with its glucosyl donor substrate, uridine 5'-diphosphoglucose (UDP-Glc), to produce the scission of the covalent bond between the terminal phosphate oxygen of UDP and the sugar ring. X-ray diffraction analysis of the PaGS/UDP-Glc complex shows no electronic density attributable to the UDP moiety, but establishes the presence in the active site of the enzyme of a glucose-like derivative that lacks the exocyclic oxygen attached to the anomeric carbon. Chemical derivatization followed by gas chromatography/mass spectrometry of the isolated glucose-like species allowed us to identify the molecule found in the catalytic site of PaGS as 1,5-anhydro-D-arabino-hex-1-enitol (AA) or its tautomeric form, 1,5-anhydro-D-fructose. These findings are consistent with a stepwise S(N) i-like mechanism as the modus operandi of retaining GTs, a mechanism that involves the discrete existence of an oxocarbenium intermediate. Even in the absence of a glucosyl acceptor, glycogen synthase (GS) promotes the formation of the cationic intermediate, which, by eliminating the proton of the adjacent C2 carbon atom, yields AA. Alternatively, these observations could be interpreted assuming that AA is a true intermediate in the reaction pathway of GS and that this enzyme operates through an elimination/addition mechanism., (Copyright © 2012 Wiley Periodicals, Inc.)

The Slater-Bonner method which is used for graphic determination of substrate constant (Ks) by linear dependence of apparent Michaelis constant (Km(app)) on the limiting rate (V(app)) of enzyme-catalysed reactions with activator participation has been critically analysed. It has been shown that although it is possible to record the mechanisms of such reactions as a scheme similar to Michaelis-Menten model which allow to find correlation Km(app) and V(app) as equation Km(app) = Ks + V(app)/k1[E]0 ([E]0 is a total enzyme concentration, k1 is a rate constant of enzyme-substrate complex formation from free enzyme and substrate) in order to calculate Ks and individual rate constants (k1, k(-1)), but this approach for investigation of all reactions with activator participation ought not to be used. The above equation is not obeyed in general, it may be true for some mechanisms only or under certain ratios of kinetic parameters of enzyme-catalysed reactions.

The comparison of volumes of cells and subcellular structures with the pH values reported for them leads to a conflict with the definition of the pH scale. The pH scale is based on the ionic product of water, K(w) = [H(+)]×[OH(-)].We used K(w) [in a reversed way] to calculate the number of undissociated H(2)O molecules required by this equilibrium constant to yield at least one of its daughter ions, H(+) or OH(-) at a given pH. In this way we obtained a formula that relates pH to the minimal volume V(pH) required to provide a physical meaning to K(w), V(pH)=10(pH-pK(w/2) x 10(pK(w/2)/N(A) (where N(A) is Avogadro's number). For example, at pH 7 (neutral at 25°C) V(pH) =16.6 aL. Any deviation from neutral pH results in a larger V(pH) value. Our results indicate that many subcellular structures, including coated vesicles and lysosomes, are too small to contain free H(+) ions at equilibrium, thus the definition of pH based on K(w) is no longer valid. Larger subcellular structures, such as mitochondria, apparently contain only a few free H(+) ions. These results indicate that pH fails to describe intracellular conditions, and that water appears to be dissociated too weakly to provide free H(+) ions as a general source for biochemical reactions. Consequences of this finding are discussed.

The biochemist and biophysicist Leonor Michaelis (1875-1949) was a renowned pioneer who worked at the interface of physical chemistry and biochemistry. He is best known for his work on the physical chemistry of proteins and enzymes and for the mathematical derivation, together with Maud Menten, of the affinity constant of the enzyme substrate bond, now known as the Michaelis-Menten constant. His thorough experimentation and careful theorizing made him critical of his contemporaries in medical biochemistry, whose work did not withstand scrutiny. His strong influence resulted from combining new concepts and approaches with traditional ones, thus bridging conceptual gaps. Most importantly, his success was brought about because he combined a critical and sharp mind with competence, passion, and determination. A review of Michaelis's approach and achievements shows that critical theory testing, as suggested by Popper, cannot explain scientific advance if taken alone; the existence of a passionate commitment to scientific beliefs, as emphasized by Polanyi, is another necessary prerequisite for the development of science.

T4 DNA ligase catalyzes phosphodiester bond formation between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA in three steps: 1) enzyme-adenylylate formation by reaction with ATP; 2) adenylyl transfer to a 5'-phosphorylated polynucleotide to generate adenylylated DNA; and 3) phosphodiester bond formation with release of AMP. This investigation used synthetic, nicked DNA substrates possessing either a 5'-phosphate or a 5'-adenylyl phosphate. Steady state experiments with a nicked substrate containing juxtaposed dC and 5'-phosphorylated dT deoxynucleotides (substrate 1) yielded kcat and kcat/Km values of 0.4±0.1 s(-1) and 150±50 μm(-1) s(-1), respectively. Under identical reaction conditions, turnover of an adenylylated version of this substrate (substrate 1A) yielded kcat and kcat/Km values of 0.64±0.08 s(-1) and 240±40 μm(-1) s(-1). Single turnover experiments utilizing substrate 1 gave fits for the forward rates of Step 2 (k2) and Step 3 (k3) of 5.3 and 38 s(-1), respectively, with the slowest step ∼10-fold faster than the rate of turnover seen under steady state conditions. Single turnover experiments with substrate 1A produced a Step 3 forward rate constant of 4.3 s(-1), also faster than the turnover rate of 1A. Enzyme self-adenylylation was confirmed to also occur on a fast time scale (∼6 s(-1)), indicating that the rate-limiting step for T4 DNA ligase nick sealing is not a chemical step but rather is most likely product release. Pre-steady state reactions displayed a clear burst phase, consistent with this conclusion.

Differences between both true and apparent rate constants and Michaelis constants have been examined. Rate constants of elementary stages of real mechanisms are true ones. True Michaelis constant Km is expressed by equation Km = (k(-1) + k2)/k. True constants may be determined for reliable mechanism only for which the equation of initial rate was obtained which displays physical sense of these constants and permits to find the method of their calculation. The true constant values are independent of concentration of reactants, activators, inhibitors, extraneous agents and pH. The apparent rate constants are such constants of the composite reaction which are observed when this reaction is described by the equation of simple reaction. Michaelis constant calculated by a half of the ultimate constant is an apparent constant. The apparent constants may be functions of several true rate constants and/or concentrations of reacting substances. The evident physical sense of apparent constants being absent, only formal relation between the reaction rate and reactant concentration independent of the investigated mechanism is provided.

Research on prion protein (PrP) and pathogenic prion has been very intensive because of its importance as model system for neurodegenerative diseases. One important aspect of this research has been the application of chemical biology tools. In this review we describe new developments like native chemical ligation (NCL) and expressed protein ligation (EPL) for the synthesis and semisynthesis of proteins in general and PrP in particular. These techniques allow the synthesis of designed tailor made analogs which can be used in conjunction with modern biophysical methods like fluorescence spectroscopy, solid state Nuclear Magnetic Resonance (ssNMR), and Electron Paramagnetic Resonance (EPR). Another aspect of prion research is concerned with the interaction of PrP with small organic molecules and metals. The results are critically reviewed and put into perspective of their implication for PrP function.

The influence of additives on the molecular dynamics of the phospholipid dimyristoylphosphatidylcholine (DMPC) in its fully hydrated liquid crystalline phase was studied. Quasielastic neutron scattering (QENS) was used to detect motions with dimensions of some Ångstroms on two different time scales, namely 60ps and 900ps. The effects of myristic acid, farnesol, cholesterol, and sodium glycocholate could consistently be explained on the basis of collective, flow-like motions of the phospholipid molecules. The influence of the additives on these motions was explained by packing effects, corresponding to the reduction of free volume. Cholesterol was found to decrease the mobility of DMPC seen on the 900ps time scale with increasing cholesterol content. In contrast, all other studied additives have no significant effect on the mobility., (Copyright © 2010 Elsevier B.V. All rights reserved.)

With this article, we wish to give an overview of our main research activities assessing the potential of a suitable polymer modification of DNA fragments to self-assemble biologically active nanostructures. Specifically, the grafting of a hydrophobic polymer segment on DNA fragments results in amphiphilic nucleotide-based macromolecules, which, owing to both chemical and physical incompatibility, organize in self-assembled structures either on surfaces or in aqueous solution. Through the combination of the existing know-how in polymer chemistry with modern analytical techniques, we are currently focusing on establishing the mechanism of self-assembly of the polymer-modified nucleotide sequences in solution and on surfaces prior to the assessment of their hybridization capacity once involved in the ensemble. With the evaluation of the potential of the functional nanostructures to undergo biological-like adhesion through hybridization one can eventually foresee that the optimal functionality of these bio-inspired systems could be fine-tuned for biological applications such as drug delivery, gene therapy, tissue engineering and the design of either biomedical devices or biosensors.

The analysis of amyloidogenic systems reveals the appearance of distinct states of aggregation for amyloid fibrils. For different proteins and under specific experimental conditions, amyloid spherulites are recognized as a significant component occurring in several protein model systems used for in vitro fibrillation studies. In this work we have developed an approach to characterize solutions containing a mixture of amyloid spherulites and individual fibrils. Using bovine insulin as the model system, sedimentation kinetics for the amyloid aggregates were followed using a combination of UV-Vis spectroscopy and cross-polarized optical microscopy. Spherulites were identified as the species undergoing sedimentation. A simple mathematical approach allows the description of the kinetics in terms of decay time/rate distribution. Moreover, based on the sedimentation kinetics, a rough estimate of the balance between amyloid spherulites and individual fibrils can be provided. Fitting the experimental data with the proposed physico-chemical approach shows self-consistent results in reasonable agreement with quantitative imaging analysis previously reported. Our results provide new physical insights into the analysis of amyloidogenic systems, providing a method to characterize the heterogeneous distribution of amyloid spherulites and simultaneously distinguish spherulites and free fibril populations. Importantly, the method can be generally applied to the characterization of polydisperse solutions containing optically traceable spherical particles in the micrometric range.

The effects of femtosecond laser ablation on the physical and chemical properties at the surface of poly methylmethacrylate (PMMA) were studied. Femtosecond laser microfabrication caused the initially wetting behavior of PMMA to become nonwetting, mainly because of the laser-induced surface porosity at the submicroscale. Static and dynamic contact angle measurements along with morphological characterization revealed that after the laser irradiation, the system lies in an intermediate regime between those theorized by Wenzel and Cassie-Baxter. Spectroscopic analysis did not evidence any significant variation in the chemical properties of the processed polymeric surfaces.

The ability to genetically encode unnatural amino acids beyond the common 20 has allowed unprecedented control over the chemical structures of recombinantly expressed proteins. Orthogonal aminoacyl-tRNA synthetase/tRNA pairs have been used together with nonsense, rare, or 4-bp codons to incorporate >50 unnatural amino acids into proteins in Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, and mammalian cell lines. This has allowed the expression of proteins containing amino acids with novel side chains, including fluorophores, post-translational modifications, metal ion chelators, photocaged and photocross-linking moieties, uniquely reactive functional groups, and NMR, IR, and x-ray crystallographic probes.

Transfer of hydrogen as a proton, hydride or hydrogen atom is an important step in many enzymic reactions. Experiments show kinetic isotope effects (KIEs) for some enzyme-catalysed hydrogen transfer reactions that deviate significantly from the limits imposed by considering the differences in mass of the isotopes alone (i.e. the semiclassical limit). These KIEs can be explained if the transfer of the hydrogen species occurs via a quantum mechanical tunnelling mechanism. The unusual temperature dependence of some KIEs has led to suggestions that enzymes have evolved to promote tunnelling through dynamics - a highly controversial hypothesis. Molecular simulations have a vital role in resolving these questions, providing a level of detail of analysis not possible through experiments alone. Here, we review computational molecular modelling studies of quantum tunnelling in enzymes, in particular focusing on the enzymes soybean lipoxygenase-1 (SLO-1), dihydrofolate reductase (DHFR), methylamine dehydrogenase (MADH) and aromatic amine dehydrogenase (AADH) to illustrate the current controversy regarding the importance of quantum effects in enzyme catalysis.

The chorismate to prephenate enzyme catalyzed reaction has been used in this review as the conduit to show different theoretical approaches that have been used over the years in our laboratory to explain its molecular mechanism. This pericyclic reaction has the advantage that other protein scaffolds such as catalytic antibodies or some promiscuous enzymes present certain chorismate mutase activity. The obtained results on all these protein environments, by comparison with the uncatalyzed reaction in solution, have been used to propose, as a general conclusion, that the origin of enzyme catalysis is in the relative electrostatic stabilization of the transition state with respect to the Michaelis complex. This feature implies that reactants of catalyzed reaction were closer to the transition state than those of the non-catalyzed reaction. From this hypothesis, and considering the features of the wild type chorismate mutases as the optimal catalyst for the reaction, some mutations on both kinds of alternative proteins have been proposed which would presumably enhance the rate constant of the chemical step.The studies presented in this paper demonstrate that the improvements and developments of the methods and techniques of theoretical and computational chemistry are now mature enough to model physic-chemical properties of biological systems with good accuracy. The combination of a potent computational protocol with molecular engineering techniques can be a promising methodology to develop novel enzymes with new or more efficient catalytic functions.

An ability to measure the biochemical parameters and structures of protein complexes at defined locations within the cellular environment would improve our understanding of cellular function. We describe widely applicable, calibrated Förster resonance energy transfer methods that quantify structural and biochemical parameters for interaction of the human estrogen receptor alpha-isoform (ER alpha) with the receptor interacting domains (RIDs) of three cofactors (SRC1, SRC2, SRC3) in living cells. The interactions of ER alpha with all three SRC-RIDs, measured throughout the cell nucleus, transitioned from structurally similar, high affinity complexes containing two ER alphas at low free SRC-RID concentrations (<2 nm) to lower affinity complexes with an ER alpha monomer at higher SRC-RID concentrations (approximately 10 nm). The methods also showed that only a subpopulation of ER alpha was available to form complexes with the SRC-RIDs in the cell. These methods represent a template for extracting unprecedented details of the biochemistry and structure of any complex that is capable of being measured by Förster resonance energy transfer in the cellular environment.

The interdisciplinary projects in bioinorganic and bioorganic chemistry of the Department of Chemistry, University of Basel led to the preparation of new systems that mimic biologically important processes and to the discovery of compounds from natural sources which are very promising with respect to medical applications. The advances in these areas are reported here.

Biophysical chemistry at the Department of Chemistry, University of Basel, covers the NMR analysis of protein-protein interaction using paramagnetic tags and sophisticated microscopy techniques investigating the dynamics of biological matter.

My career has been focused in two major areas, analytical chemistry and biochemistry of complex lipids and glycoconjugates. Included here are the pioneering work on the gas chromatography of long-chain sphingolipid bases, carbohydrates, steroids and urinary organic acids. Mass spectrometry was utilized extensively in structural studies of sphingolipids, fatty acids, carbohydrates, steroids, urinary organic acids, polyisoprenoid alcohols, and juvenile hormone. Computer systems were developed for the acquisition and analysis of mass spectra, and were used for development of automated metabolic profiling of complex mixtures of metabolites. Fabry's disease was discovered to be a glycosphingolipidosis. Enzymes of lysosomal metabolism of glycosphingolipids were purified, characterized, and used in one of the first demonstrations of the feasibility of enzyme replacement therapy in a lysosomal storage disorder (Fabry's disease). Extracellular sialidases were studied to evaluate the hypothesis that they might be involved in the regulation of membrane growth factor receptors. The enzyme for hematoside synthesis was purified and characterized.

This paper presents a methodology for translating iterative arithmetic computation, specified as high-level programming constructs, into biochemical reactions. From an input/output specification, we generate biochemical reactions that produce output quantities of proteins as a function of input quantities performing operations such as addition, subtraction, and scalar multiplication. Iterative constructs such as "while" loops and "for" loops are implemented by transferring quantities between protein types, based on a clocking mechanism. Synthesis first is performed at a conceptual level, in terms of abstract biochemical reactions - a task analogous to high-level program compilation. Then the results are mapped onto specific biochemical reactions selected from libraries - a task analogous to machine language compilation. We demonstrate our approach through the compilation of a variety of standard iterative functions: multiplication, exponentiation, discrete logarithms, raising to a power, and linear transforms on time series. The designs are validated through transient stochastic simulation of the chemical kinetics. We are exploring DNA-based computation via strand displacement as a possible experimental chassis.

The pathway by which ubiquitin chains are generated on substrate through a cascade of enzymes consisting of an E1, E2 and E3 remains unclear. Multiple distinct models involving chain assembly on E2 or substrate have been proposed. However, the speed and complexity of the reaction have precluded direct experimental tests to distinguish between potential pathways. Here we introduce new theoretical and experimental methodologies to address both limitations. A quantitative framework based on product distribution predicts that the really interesting new gene (RING) E3 enzymes SCF(Cdc4) and SCF(beta-TrCP) work with the E2 Cdc34 to build polyubiquitin chains on substrates by sequential transfers of single ubiquitins. Measurements with millisecond time resolution directly demonstrate that substrate polyubiquitylation proceeds sequentially. Our results present an unprecedented glimpse into the mechanism of RING ubiquitin ligases and illuminate the quantitative parameters that underlie the rate and pattern of ubiquitin chain assembly.

To determine the levels of post-translational modifications, we needed a quantitative technique that would allow comparison of the amounts of acetylated versus mono-, di-, and tri-methylated lysines in histones. One method, IVICAT, generates trimethyl-amines and could be used, but is technically challenging. We have modified this technique to be used with standard laboratory equipment so that this chemistry is accessible to most proteomics laboratories.

Knowledge of the existence and structure of intermediates on the reaction pathway is necessary before specific details of the mechanism may be successfully resolved. However, enzymatic catalysis is an extremely fast process. This rapidity of enzyme-catalyzed reactions and the short life times of intermediates represent a major problem in studying the dynamic processes which occur during catalysis, as they prevent the accumulation of intermediates under normal conditions for concentrations and time periods required by most high-resolution structural methods. Therefore, a method that would utilize specific substrates but would permit the detection and characterization of intermediates was highly desired. As one of the approaches to overcome this problem the use of cryoenzymology to allow the accumulation and stabilization of intermediates at very low temperatures was proposed. This review describes the contribution of Prof. Anthony L. Fink to cryoenzymology and shows how his work shaped this exciting area.

The uncertainty of measurement (UM) or measurement uncertainty is known as the parameter associated with the result of a measurement. Repeated measurements usually reveal slightly different results for the same analyte, sometimes a little higher, sometimes a little lower, because the results of a measurement are depending not only by the analyte itself, but also, by a number of error factors that could give doubts about the estimate. The uncertainty of the measurement represent the quantitative, mathematically expression of this doubt. UM is a range of measured values which is probably to enclose the true value of the measured. Calculation of UM for all types of laboratories is regularized by the ISO Guide to the Expression of Uncertainty in Measurement (abbreviated GUM) and the SR ENV 13005 : 2003 (both recognized by European Accreditation). Even if the GUM rules about UM estimation are very strictly, the offering of the result together with UM will increase the confidence of customers (patients or physicians). In this study the authors are presenting the possibilities of UM assessing in labs from our country by using the data obtained in the procedures of methods validation, during the internal and external quality control.

Psychosine is an important bioactive sphingolipid metabolite and plays an essential role in the pathogenesis of Krabbe's disease. Herein, we extended shotgun lipidomics for the characterization and quantitation of psychosine in alkaline-treated crude lipid extracts by using neutral loss scan of 180 amicro (i.e., galactose) in the positive-ion mode. Specifically, we semi-synthesized N,N-dimethyl psychosine and used it as an internal standard for quantitation of psychosine. After characterization of the fragmentation patterns of psychosine and the selected internal standard and optimization of the experimental conditions, we demonstrated that a broad linear dynamic range for the quantitation of psychosine and a limit of detection at a concentration of low fmol/microl were achieved using this approach. The developed method is generally simpler and more efficient than other previously reported methods. Multiple factors influencing quantitation of psychosine were extensively examined and/or discussed. The levels of psychosine in diabetic mouse nerve tissue samples were determined by the developed methodology. Collectively, the developed approach, as a new addition to the shotgun lipidomics technology, will be extremely useful for understanding the pathways/networks of sphingolipid metabolism and for exploring the important roles of psychosine in a variety of physiological and pathological conditions.

Depending on the method of deposition, reactive sites of polysaccharides on substrates may not be available when their reducing ends have been used to covalently bind them to the substrates. Here we present a method that allows surface density measurements of reducing-end covalently bound polysaccharides in a procedure that cleaves the polysaccharide chain from the surface via hydrazinolysis and deamination, leaving on the surface a disaccharide that is later radiolabeled with an aldehyde in a reaction with enamine formation. The method described has the advantage that it may be used with any polysaccharide patterned to any surface exposing an amino-terminated monolayer by reductive amination of their galactosamine or glucosamine repeating units. We illustrate the technique with the quantitation of glycosaminoglycans (GAGs) on silanized glass surfaces.

A simple kinetic model is developed to describe the dynamic behavior of myeloma cell growth and cell metabolism. Glucose, glutamine as well as lysine are considered as growth limiting substrates. The cell growth was restricted as soon as the extracellular lysine is exhausted and then intracellular lysine becomes a growth limiting substrate. In addition, a metabolic regulator model together with the Monod model is used to deal with the growth lag phase after inoculation or feeding. By using these models, concentrations of substrates and metabolites, as well as densities of viable and dead cells are quantitatively described. One batch cultivation and two fed-batch cultivations with pulse feeding of nutrients are used to validate the model.

A one-pot biotransformation for the generation of a series of L-aminotryptophans using a readily prepared protein extract containing tryptophan synthase is reported. The extract exhibits remarkable stability upon freeze-drying, and may be stored and used for long periods after its preparation without significant loss of activity.

Just as complex electronic circuits are built from simple Boolean gates, diverse biological functions, including signal transduction, differentiation, and stress response, frequently use biochemical switches as a functional module. A relatively small number of such switches have been described in the literature, and these exhibit considerable diversity in chemical topology. We asked if biochemical switches are indeed rare and if there are common chemical motifs and family relationships among such switches. We performed a systematic exploration of chemical reaction space by generating all possible stoichiometrically valid chemical configurations up to 3 molecules and 6 reactions and up to 4 molecules and 3 reactions. We used Monte Carlo sampling of parameter space for each such configuration to generate specific models and checked each model for switching properties. We found nearly 4,500 reaction topologies, or about 10% of our tested configurations, that demonstrate switching behavior. Commonly accepted topological features such as feedback were poor predictors of bistability, and we identified new reaction motifs that were likely to be found in switches. Furthermore, the discovered switches were related in that most of the larger configurations were derived from smaller ones by addition of one or more reactions. To explore even larger configurations, we developed two tools: the "bistabilizer," which converts almost-bistable systems into bistable ones, and frequent motif mining, which helps rank untested configurations. Both of these tools increased the coverage of our library of bistable systems. Thus, our systematic exploration of chemical reaction space has produced a valuable resource for investigating the key signaling motif of bistability.