ChemBioChem

Real‐time PCR is the state‐of‐the‐art technique to quantify nucleic acids for mutation detection, genotyping and chimerism analysis. Since its development in the 1990s, many different assay formats have been developed and the number of real‐time PCR machines of different design is continuously increasing. This review provides a survey of the instruments and assay formats available and discusses the pros and cons of each. The principles of quantitative real‐time PCR and melting curve analysis are explained. The quantification algorithms with internal and external standardization are derived mathematically, and potential pitfalls for the data analysis are discussed. Finally, examples of applications of this extremely versatile technique are given that demonstrate the enormous impact of real‐time PCR on life sciences and molecular medicine.

The initial transition of amyloid β (1–42) (Aβ42) soluble monomers/small oligomers from unordered/α‐helix to a β‐sheet‐rich conformation represents a suitable target to design new potent inhibitors and to obtain effective therapeutics for Alzheimer's disease. Under optimized conditions, this reliable and reproducible CD kinetic study showed a three‐step sigmoid profile that was characterized by a lag phase (prevailing unordered/α‐helix conformation), an exponential growth phase (increasing β‐sheet secondary structure) and a plateau phase (prevailing β‐sheet secondary structure). This kinetic analysis brought insight into the inhibitors' mechanism of action. In fact, an increase in the duration of the lag phase can be related to the formation of an inhibitor–Aβ complex, in which the non‐amyloidogenic conformation is stabilized. When the exponential rate is affected exclusively, such as in the case of Congo red and tetracycline, then the inhibitor affinity might be higher for the pleated β‐sheet structure. Finally, by adding the inhibitor at the end of the exponential phase, the soluble protofibrils can be disrupted and the Aβ amyloidogenic structure can revert into monomers/small oligomers. Congo red and tetracycline preferentially bind to amyloid in the β‐sheet conformation because both decreased the slope of the exponential growth, even if to a different extent, whereas no effect was observed for tacrine and galantamine. Some very preliminary indications can be derived about the structural requirements for binding to nonamyloidogenic or β‐sheet amyloid secondary structure for the development of potent antiaggregating agents. On these premises, memoquin, a multifunctional molecule that was designed to become a drug candidate for the treatment of Alzheimer's disease, was investigated under the reported circular dichroism assay and its anti‐amyloidogenic mechanism of action was elucidated.

This article deals with the solution structure determination of paramagnetic metalloproteins by NMR spectroscopy. These proteins were believed not to be suitable for NMR investigations for structure determination until a decade ago, but eventually novel experiments and software protocols were developed, with the aim of making the approach suitable for the goal and as user‐friendly and safe as possible. In the article, we also give hints for the optimization of experiments with respect to each particular metal ion, with the aim of also providing a handy tool for nonspecialists. Finally, a section is dedicated to the significant progress made on ¹³C direct detection, which reduces the negative effects of paramagnetism and may constitute a new chapter in the whole field of NMR spectroscopy.

Enzymes have the potential to catalyse a wide variety of chemical reactions. They are increasingly being sought as environmentally friendly and cost‐effective alternatives to conventional catalysts used in industries ranging from bioremediation to applications in medicine and pharmaceutics. Despite the benefits, they are not without their limitations. Many naturally occurring enzymes are not suitable for use outside of their native cellular environments. However, protein engineering can be used to generate enzymes tailored for specific industrial applications. Directed evolution is particularly useful and can be employed even when lack of structural information impedes the use of rational design. The aim of this review is to provide an overview of current industrial applications of enzyme technology and to show how directed evolution can be used to modify and to enhance enzyme properties. This includes a brief discussion on library generation and a more detailed focus on library screening methods, which are critical to any directed evolution experiment.

Lipopolysaccharides (LPS) are cell wall components of Gram‐negative bacteria. These molecules behave as bacterial endotoxins and their release into the bloodstream is a determinant of the development of a wide range of pathologies. These amphipathic molecules can self‐aggregate into supramolecular structures with different shapes and sizes. The formation of these structures occurs when the LPS concentration is higher than the apparent critical micelle concentration (CMC_a). Light scattering spectroscopy (both static and dynamic) was used to directly characterize the aggregation process of LPS from Escherichia coli serotype 026:B6. The results point to a CMC_a value of 14 μg mL⁻¹ and the existence of premicelle LPS oligomers below this concentration. Both structures were characterized in terms of molecular weight (5.5×10⁶ and 16×10⁶ g mol⁻¹ below and above the CMC_a, respectively), interaction with the aqueous environment, gyration radius (56 and 105 nm), hydrodynamic radius, (60 and 95 nm) and geometry of the supramolecular structures (nearly spherical). Our data indicates that future in vitro experiments should be carried out both below and above the CMC_a. The search for drugs that interact with the aggregates, and thus change the CMC_a and condition LPS interactions in the bloodstream, could be a new way to prevent certain bacterial‐endotoxin‐related pathologies.

A mirror image phage display approach was used to identify novel and highly specific ligands for Alzheimer's disease amyloid peptide Aβ(1–42). A randomized 12‐mer peptide library presented on M13 phages was screened for peptides with binding affinity for the mirror image of Aβ(1–42). After four rounds of selection and amplification the peptides were enriched with a dominating consensus sequence. The mirror image of the most representative peptide (D‐pep) was shown to bind Aβ(1–42) with a dissociation constant in the submicromolar range. Furthermore, in brain tissue sections derived from patients that suffered from Alzheimer's disease, amyloid plaques and leptomeningeal vessels containing Aβ amyloid were stained specifically with a fluorescence‐labeled derivative of D‐pep. Fibrillar deposits derived from other amyloidosis were not labeled by D‐pep. Possible applications of this novel and highly specific Aβ ligand in diagnosis and therapy of Alzheimer's disease are discussed.

Exosomes and microvesicles are two classes of submicroscopic vesicle released by cells into the extracellular space. Collectively referred to as extracellular vesicles, these membrane containers facilitate important cell–cell communication by carrying a diverse array of signaling molecules, including nucleic acids, proteins, and lipids. Recently, the role of extracellular vesicle signaling in cancer progression has become a topic of significant interest. Methods to detect and target exosomes and microvesicles are needed to realize applications of extracellular vesicles as biomarkers and, perhaps, therapeutic targets. Detection of exosomes and microvesicles is a complex problem as they are both submicroscopic and of heterogeneous cellular origins. In this Minireview, we highlight the basic biology of extracellular vesicles, and address available biochemical and biophysical detection methods. Detectible characteristics described here include lipid and protein composition, and physical properties such as the vesicle membrane shape and diffusion coefficient. In particular, we propose that detection of exosome and microvesicle membrane curvature with lipid chemical probes that sense membrane shape is a distinctly promising method for identifying and targeting these vesicles.

Pichia pastoris is a well‐known platform strain for heterologous protein expression. Over the past five years, different strategies to improve the efficiency of recombinant protein expression by this yeast strain have been developed; these include a patent‐free protein expression kit, construction of the P. pastoris CBS7435Ku70 platform strain with its high efficiency in site‐specific recombination of plasmid DNA into the genomic DNA, the design of synthetic promoters and their variants by combining different core promoters with multiple putative transcription factors, the generation of mutant GAP promoter variants with various promoter strengths, codon optimization, engineering the α‐factor signal sequence by replacing the native glutamic acid at the Kex2 cleavage site with the other 19 natural amino acids and the addition of mammalian signal sequence to the yeast signal sequence, and the co‐expression of single chaperones, multiple chaperones or helper proteins that aid in recombinant protein folding. Publically available high‐quality genome data from multiple strains of P. pastoris GS115, DSMZ 70382, and CBS7435 and the continuous development of yeast expression kits have successfully promoted the metabolic engineering of this strain to produce carotenoids, xanthophylls, nootkatone, ricinoleic acid, dammarenediol‐II, and hyaluronic acid. The cell‐surface display of enzymes has obviously increased enzyme stability, and high‐level intracellular expression of acyl‐CoA and ethanol O‐acyltransferase, lipase and d‐amino acid oxidase has opened up applications in whole‐cell biocatalysis for producing flavor molecules and biodiesel, as well as the deracemization of racemic amino acids. High‐level expression of various food‐grade enzymes, cellulases, and hemicellulases for applications in the food, feed and biorefinery industries is in its infancy and needs strengthening.

Monooxygenase mutants: A minimal and highly enriched CYP102A1 mutant library was constructed by combining five hydrophobic amino acids in two positions. The library was screened with four different terpene substrates. Eleven variants demonstrated either a strong shift or improved regio‐ or stereoselectivity during oxidation of at least one substrate as compared to CYP102A1 wild type.magnified image

A minimal CYP102A1 mutant library of only 24 variants plus wild type was constructed by combining five hydrophobic amino acids (alanine, valine, phenylalanine, leucine and isoleucine) in two positions. Both positions are located close to the centre of the haem group. The first, position 87, has been shown to mediate substrate specificity and regioselectivity in CYP102A1. The second hotspot, position 328, was predicted to interact with all substrates during oxidation and has previously been identified by systematic analysis of 31 crystal structures and 6300 sequences of cytochrome P450 monooxygenases. By systematically altering the size of the side chains, a broad range of binding site shapes was generated. All variants were functionally expressed in E. coli. The library was screened with four terpene substrates geranylacetone, nerylacetone, (4R)‐limonene and (+)‐valencene. Only three variants showed no activity towards all four terpenes, while eleven variants demonstrated either a strong shift or improved regio‐ or stereoselectivity during oxidation of at least one substrate as compared to CYP102A1 wild type.