Glycine-rich proteins

Glycine-rich proteins (GRPs) containing more than 60% glycine have been found in different tissues from many eukaryotic species. Despite the availability of literature on different groups of GRPs, there are few reports in which they are all considered and compared together. Some of these proteins are components of the cell walls of many higher plants. In most cases, it has been shown that they are accumulated in the vascular tissues and that their synthesis is part of the plant’s defense mechanism. Other distinct types of GRPs are characterized by having structures and functions similar to animal cytokeratins or by a domain with typical RNA-binding motifs. The availability of cloned GRP genes facilitates the study of the function of this diverse class of proteins, which is expected to enhance the understanding of cell physiology.     

Two-dimensional electrophoresis of membrane proteins: separation of myelin proteins

A method for two-dimensional electrophoretic separation of myelin proteins is presented. The first dimension consists of isoelectric focusing of lyophilized and delipidated membrane proteins, solubilized in a mixture of the nonionic detergent Triton X-100, the zwitterionic detergent CHAPS, 9 M urea and carrier ampholytes, and incorporated into a slab gel before separation. Subsequent discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed by moulding the isoelectric focusing slab gel with its supporting glass plate into the stacking gel. This method proved to give highly reproducible results since mechanical forces and thus the risk of stretching, folding or rupture of the isoelectric focusing slab gel is minimized. Furthermore, by immunoblotting, the positions of myelin-associated glycoprotein and 2',3'-cyclic nucleotide 3'-phosphodiesterase were established with specific antibodies.     

Isotachophoresis of proteins

The analytical separation of proteins by isotachophoresis (ITP) was achieved in a short electrophoretic path and with a resolution comparable to that of isoelectric focusing by the appropriate selection of (1) a mixture of ampholytes as spacers to generate linear gradients of electrophoretic mobility and (2) the counter ions chosen to buffer the complete pH gradient generated. This ITP technique is exemplified by the analysis of plasma proteins in agarose gels. Up to 46 samples in the same gel plate were analysed. The resolution was such that at least 30 clear and discrete bands per sample could be observed after staining with Coomassie Brilliant Blue. The resolving power of ITP could be further increased for the study of a particular protein or zone by the selection of suitable spacers and counter ions.     

Monolayers of proteins

Summary Monolayers of various proteins were studied at airwater interface. An equation of state of the virial type, as developed byM. L. Huggins, was applied to the surface pressure-area measurements. It was found that theHuggins equation based on statistical thermodynamics was applicable to the proteins. From this treatment of the results, the degree of submersion of protein could be determined, which was found to be dependent on the degree of unfolding of the protein at the interface.     

Point-of-care testing of proteins

Point-of-care testing (POCT) is a fast developing area in clinical diagnostics that is considered to be one of the main driving forces for the future in vitro diagnostic market. POCT means decentralized testing at the site of patient care. The most important POCT devices are handheld blood glucose sensors. In some of these sensors, after the application of less than 1 μl whole blood, the results are displayed in less than 10 s. For protein determination, the most commonly used devices are based on lateral flow technology. Although these devices are convenient to use, the results are often only qualitative or semiquantitative. The review will illuminate some of the current methods employed in POCT for proteins and will discuss the outlook for techniques (e.g., electrochemical immunosensors) that could have a great impact on future POCT of proteins.     

Melting domain in proteins

Recent progress in thermodynamic aspects of proteins, free or immobilized on solid support, are described. In agreement with results observed with Ribonuclease A [9], DSC analysis on α-chymotrypsin confirms a decoupling of melting domains with the immobilized protein in a large range ofpH.     

朊病毒蛋白prion的研究进展

传染性海绵状脑病是一类致死性的神经系统退行性疾病,其发病机制与朊病毒蛋白prion的异常构象有关.迄今为止,prion致病的构象变化机制依然是一个未解之谜.国内外研究人员从不同切入点着手,采用物理、化学、生物等多种学科的技术手段,发现并积累了大量与prion构象转变相关的有价值的信息.本文综述了近年来人们在prion蛋白的三维结构、动力学、去折叠以及相互作用等方面取得的研究进展,并简要地介绍了本实验室在兔prion蛋白溶液结构和动力学方面的研究工作.     

Oriented immobilization of proteins

Immobilized enzymes have found numerous applications in analytical, clinical, environmental and industrial chemistry. However, in most cases, immobilization leads to partial or total loss of activity. It is widely believed that the loss in activity is due to attachment of proteins on the immobilization support through several amino acid residues. This results in a random orientation of the immobilized protein and in increased structural deformation due to multi-point attachment. Several researchers have explored ways to orient proteins on surfaces, such that orderly organization, single point attachment and accessibility of the active site (or binding site) are possible. This article reviews the various approaches available to achieve oriented immobilization of proteins and its applications in several disciplines.     

Docking to heme proteins

In silico screening has become a valuable tool in drug design, but some drug targets represent real challenges for docking algorithms. This is especially true for metalloproteins, whose interactions with ligands are difficult to parametrize. Our docking algorithm, EADock, is based on the CHARMM force field, which assures a physically sound scoring function and a good transferability to a wide range of systems, but also exhibits difficulties in case of some metalloproteins. Here, we consider the therapeutically important case of heme proteins featuring an iron core at the active site. Using a standard docking protocol, where the iron–ligand interaction is underestimated, we obtained a success rate of 28% for a test set of 50 heme‐containing complexes with iron‐ligand contact. By introducing Morse‐like metal binding potentials (MMBP), which are fitted to reproduce density functional theory calculations, we are able to increase the success rate to 62%. The remaining failures are mainly due to specific ligand–water interactions in the X‐ray structures. Testing of the MMBP on a second data set of non iron binders (14 cases) demonstrates that they do not introduce a spurious bias towards metal binding, which suggests that they may reliably be used also for cross‐docking studies. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2009     

Isoelectric focusing of membrane proteins: high resolution separation of myelin proteins

A mixture of the nonionic detergent Triton X-100, the zwitterionic detergent 3-[(cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS), 9M urea and carrier ampholytes was found comparable to media containing sodium dodecyl sulfate in the capacity for solubilization of myelin proteins, including the highly hydrophobic proteolipid protein. The solubilized sample was incorporated into the polymerization mixture before moulding an ultrathin gel, with heat convection characteristics allowing a high wattage to be applied, thus allowing fast separation with high resolving power. Since the most basic protein in myelin focuses at a pH greater than 10, fast separation is essential in order to minimize decay of the cathodic end of the pH gradient.     

Two-photon excitation of proteins

Fluorescence emission after two-photon excitation at 532 nm by means of a Nd : YAG laser is observed in apohemoglobin, hemoglobin, albumin and tryptophan at room temperature. The experimental results show that the fluorescence of these proteins originates from tryptophan residues. No fluorescence of a biphotonic nature could be detected from lysozyme and tyrosine.     

Spread monolayers of proteins

The study of spread monolayers of proteins is of interest for understanding the fundamental behavior of proteins as well as the many phenomena resulting from their ubiquitous presence at interfaces in nature. Spread monolayers of proteins is a branch of the developing field of membrane mimetic chemistry. In recent times, it has been somewhat neglected in comparison to other branches (such as bilayers, liposomes and vesicles), despite the unique advantage that the arrangement and packing of molecules in monolayers may be measured and controlled. Methods for spreading proteins and techniques used for their manipulation are outlined. As well as the more traditional methods (such as surface pressure, potential and viscosity), more recent innovations, including removal of monolayers on slides for study by radiotracer techniques, electron diffraction and infrared (IR) spectroscopy, are discussed. Direct optical methods for the study of monolayers in situ are also available (e.g., multiple reflectance spectroscopy, ellipsometry). The use of measurements in the low pressure region to measure molecular weights is discussed. At higher pressures, configurational changes, surface coagulation and desorption are all observed. Experimental and theoretical work on the desorption of proteins from the air/water interface is reviewed. The introduction of multicompartment film balances has proved valuable for the study of reactions occurring in monolayers. This instrumentation has been applied to the study of enzyme reactions at the surface, of direct relevance to reactions where the enzyme is immobilized in the cell membrane. Some applications of monolayer studies are briefly illustrated with reference to biological membranes, foams and emulsions and biomedical problems.     

Chemical synthesis of proteins

Powerful new enzymatic and chemical methods for coupling unprotected peptide fragments are making the assembly of large synthetic proteins possible. By allowing the use of novel backbones and the incorporation of multiple unnatural amino acids at specific sites, these methods promise large expansion of the repertoire of protein molecules accessible to research.     

Creating functional artificial proteins

Much is now known about how protein folding occurs, through the sequence analysis of proteins of known folding geometry and the sequence/structural analysis of proteins and their mutants. This has allowed not only the modification of natural proteins but also the construction of de novo polypeptides with predictable folding patterns. Structure/function analysis of natural proteins is used to construct derived versions that retain a degree of biological activity. The constructed versions made of either natural or artificial sequences contain critical residues for activity such as receptor binding. In some cases, the functionality is introduced by incorporating binding sites for other elements, such as organic cofactors or transition metals, into the protein scaffold. While these modified proteins can mimic the function of natural proteins, they can also be constructed to have novel activities. Recently engineered photoactive proteins are good examples of such systems in which a light-induced electron transfer can be established in normally light-insensitive proteins. The present review covers some aspects of protein design that have been used to investigate protein receptor binding, cofactor binding and biological electron transfer.     

Mass spectrometry of proteins

Several of the present mass spectrometric techniques have sufficient mass range and sensitivity to be viable alternatives or valuable supplements to traditional methods in protein chemistry. The precision of the molecular masses determined by mass spectrometry allows highly specific protein and peptide characterization as well as identification and localization of post translational modifications. In this article the principles and practical performance of the key techniques are discussed and examples of applications given.     

Ultrasensitive assays for proteins

Proteins are essential components of organisms and are involved in a wide range of biological functions. There are increasing demands for ultra-sensitive protein detection, because many important protein biomarkers are present at ultra-low levels, especially during the early stages of disease. Measuring proteins at low levels is also crucial for investigations of the protein synthesis and functions in biological systems. In this review, we summarize the recent developments of novel technology enabling ultrasensitive protein detection. We focus on two groups of techniques that involve either polymerase amplification of affinity DNA probes or signal amplification by the use of nano-/micro-materials. The polymerase-based amplification of affinity DNA probes indirectly improves the sensitivity of protein detection by increasing the number of detection molecules. The use of nano-/micro-materials conjugated to affinity probes enhances the measurement signals by using the unique electrical, optical, and catalytic properties of these novel materials. This review describes the basic principles, performances, applications, merits, and limitations of these techniques.     

Separation of muscle proteins

This review covers various methods used in the separation and isolation of individual muscle contractile proteins. It is shown which methods have been most useful for the separation of contractile proteins and their fragments and in extending our knowledge of muscle biochemistry and physiology.