Protein Molecular Structure Research Articles

Matrix-assisted laser desorption/ionization- quadrupole ion trap-time of flight mass spectrometry sequencing resolves structures of unidentified peptides obtained by in-gel tryptic digestion of haptoglobin derivatives from human plasma proteomes.

Two-dimensional gel electrophoresis-separated and excised haptoglobin alpha2-chain protein spots were subjected to in-gel digestion with trypsin. Previously unassigned peptide ion signals observed in mass spectrometric fingerprinting experiments were sequenced using the matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight (MALDI-QIT-TOF) mass spectrometer and showed that the haptoglobin alpha-chain derivative under study was cleaved by trypsin unspecifically. Abundant cleavages occurred C-terminal to histidine residues at H23, H28, and H87. In addition, mild acidic hydrolysis leading to cleavage after aspartic acid residues at D13 was observed. The uninterpreted tandem mass spectrometry (MS/MS) spectrum of the peptide with ion signal at 2620.19 was submitted to database search and yielded the identification of the corresponding peptide sequence comprising amino acids (aa) aa65-87 from the haptoglobin alpha-chain protein. Also, the presence of a mixture of two tryptic peptides (mass to charge ratio m/z 1708.8; aa40-54, and aa99-113, respectively), that is caused by a tiny sequence variation between the two repeats in the haptoglobin alpha2-chain protein was resolved by MS/MS fragmentation using the MALDI-QIT-TOF mass spectrometer instrument. Advantageous features such as (i) easy parent ion creation, (ii) minimal sample consumption, and (iii) real collision induced dissociation conditions, were combined successfully to determine the amino acid sequences of the previously unassigned peptides. Hence, the novel mass spectrometric sequencing method applied here has proven effective for identification of distinct molecular protein structures.

Advances in care of children with hemophilia.

Care for children with severe hemophilia has moved from pediatric hospital wards and rehabilitation services to the home, school, and community. Advances in hemophilia are due largely to the development of specialized hemophilia treatment centers, which created a system of comprehensive care and focused healthcare efforts on prevention and education. Parallel advances in coagulation resulted in identification of clotting factors VIII and IX, elucidation of the protein molecular and biochemical structures and functions, sequencing of their respective genes and transfer of the human genes for production of proteins by recombinant technology, and development of gene therapy. The tragedy of the human immunodeficiency virus and hepatitis C raised awareness in patients as well as healthcare providers of the vulnerability of blood products to viral contamination and spurred progress in science leading to viral inactivation of purified proteins. Concomitantly, physicians treating bleeding episodes in the clinic investigated pharmacokinetics and pharmacoeconomics of various strategies of clotting factor replacement. The observation that trough factor levels as low as 1 to 2% were adequate to prevent most bleeding episodes led to current prophylactic regimens that allow boys to participate fully in school and community activities while factor concentrate is infused at home on a regular schedule. Currently, children with hemophilia look forward to a normal life expectancy and excellent health-related quality of life. Physician and community partnerships through research and advocacy societies have accelerated clinical advancements as well as extension of treatment to developing countries. The future of hemophilia promises a cure with gene therapy. Given the past accomplishments in hemophilia, a long-term solution to replacement of the genetically deficient protein lies on the horizon.

Spectroscopic techniques coupled with chemometric tools for structure and texture determinations in dairy products

Abstract Structure conditions both textural and flavour properties of cheeses and dairy products. Until recently, delineating the structure of the food matrices and the interactions between components in dairy products has been impaired by the lack of techniques. More and more evidence shows that front face fluorescence spectroscopy and attenuated total reflection infrared spectroscopies may be valuable techniques. These spectroscopic techniques remove methodological locks and allow the structure at molecular level and the dynamics of “real dairy products” to be investigated. Over the last few years, we gave most of our interest to the delineation of the molecular structure of dairy products and to the understanding of the relationships between structure and texture in cheese. The analysis of large spectral collections recorded for dairy products using multivariate analysis techniques such as principal component analysis, factorial discriminant analysis and canonical correlation analysis makes it possible to extract relevant information related to the molecular structure of proteins and fats in semi-hard cheeses, the interactions between dairy product components and the relation between structure and texture of soft and semi-hard cheeses. The presented data show that the texture of a dairy product is a reflection of its structure at the molecular level.

高圧力の可能性に挑む ポストゲノム時代の高圧生物化学の展望

One of the new focuses of the biological science was genomic science during the 20th century. The new frontier for the 21st century is protein science. Fundamental thermodynamic and kinetic theories, especially volume changes and activation volumes, for the pressure denaturation of proteins are described. The recent studies of molecular structure of proteins utilizing high-pressure spectroscopes (FTIR, NMR, SAXS) and the MD computer simulation are reviewed.

Capturing time-resolved changes in molecular structure by negative staining

Imaging structural intermediates of biological processes is a key step in understanding biological function. Because intermediates are commonly short-lived, lasting only milliseconds, the main methods used to capture them have been conventional imaging of analog or inhibited states, having extended lifetimes, or rapid (millisecond timescale) freezing of intermediates with subsequent observation by cryo-EM. We have developed a simpler method that fixes structure on the millisecond timescale. The procedure consists of briefly (milliseconds) exposing the macromolecular structure of interest on an EM grid to conditions that initiate the structural change, then immediately fixing with uranyl acetate or tannic acid. Specimens are then observed by negative staining. The key finding that validates this approach is our demonstration that uranyl acetate, and in some cases tannic acid, fixes protein molecular structure on the millisecond timescale. This is demonstrated by our observation that exposure of actin and myosin filaments to these fixatives for as little as 10ms is sufficient to fully preserve them against changes that normally induce rapid and major alteration in their molecular structure. Fixation appears to stabilize both ionic and hydrophobic bonds. This approach should be of general utility for studying transient molecular changes in many systems.

A thermodynamic interpretation of malignancy: do the genes come later?

Current theories on cancer development focus on ‘unlucky’ mutations affecting oncogenes or tumour suppressor genes. In this article a theory will be developed which interprets cancer as an adaptive phenomenon – a response to cellular stress induced by an energetic overload which would ultimately lead to an increase in cellular entropy. One of these adaptive mechanisms is paneuploid polyploidization, a phenomenon frequently described in malignant tumours. This inherent property of the genome to multiply with limited sequence variability may be involved just to make new proteins which are more appropriate to manage the harmful situation of energetic overload. Another important mechanism to prevent increasing entropy is the change in chirality of proteins and carbon hydrates because the use of enantiomers with higher intrinsic energy ultimately reduces entropy of the cell. These chiral alterations in turn affect the molecular structures of proteins and DNA, resulting in abnormal function of the former and disturbances of replication, transcription and repair of the latter. Moreover, the altered proteins may – as a secondary step – induce structural changes of the DNA. Because changes in chirality affect the structure of a cell randomly, one can expect alterations of multiple genes or proteins, and this is exactly what has been described in the literature. Therefore, this hypothesis may help to clarify confusing findings of tumour genetics accumulated over the last two decades. Cancer could be seen as a reaction of a cell to entrap energy which reduces entropy, or, in other words, cancer may best be regarded as entropic devolution.

Molecular structures of proteins involved in vesicle fusion.

We present a summary of the structures of 13 proteins involved in the docking and fusion of intracellular transport vesicles to their target membranes.

Molecular structures of proteins involved in vesicle coat formation.

This review includes 16 structures of vesicle coat components and accessory proteins and a description of their roles in vesicle budding or coat disassembly.

Macromolecular Crystals in the Service of Biotechnology and Medicine

ABSTRACTThe application of genetic and molecular biological methods to the engineering of proteins, the engine of the biotechnology revolution, has become increasingly dependent on knowledge of protein, nucleic acid and virus molecular structure. Structural information of high precision, essential to this enterprise, can only be obtained through X-ray crystallographic techniques. The quality of this information is directly a function of the properties and degree of perfection of the crucial intermediates, the macromolecular crystals. As a consequence, there is now broad and intense interest in developing new methods, procedures and reagents for the nucleation and growth of such crystals. There is, in addition, an expanded interest in the properties of biological crystals and the use of physical measurements in improving approaches to growing better and larger crystals. Here some of the fundamentals of macromolecular crystal growth will be reviewed, and some current trends in the field remarked upon, including the new initiative to develop a national structural genomics program.

The glutenin macropolymer of wheat flour doughs: structure–function perspectives

Gluten is the main functional component of wheat, and is the main source of the viscoelastic properties in a dough. Dough properties are primarily governed by the structure of gluten, and interactions within the protein complex. While starch and water are the main components of doughs, the physical properties of a dough arise from interactions between gluten proteins, particularly the disulfide-bonded glutenin macropolymer. The primary structures of glutenin subunits are well characterized, but information on the specificity and pattern on their interactions has been lacking, despite the fact that dough is one of the key food systems where the likely link between molecular protein structure and function is compelling. The structure of polymeric glutenin has been difficult to study since it comprises several dozen different disulfide-bonded polypeptides in the molecular mass range of millions to hundreds of millions. Recently, a range of techniques has been used to help unravel structural information. These include biochemical methods, particularly chromatography and electrophoresis systems which are suitable for examining polydisperse high molecular weight protein complexes of limited solubility; small-scale rheological testing systems, in which the function of individual components (either isolated or from expressed genes) can be determined; and a range of microscopy-based techniques, including confocal light scanning microscopy, transmission electron microscopy, and scanning electron microscopy. Utilizing these techniques, an improved understanding of the relationship between polymeric glutenin structure and dough function has been obtained. It has been proposed that glutenin subunits provide a structural backbone to the glutenin macropolymer through the formation of disulfide bonds that are highly resistant to cleavage [1]. The inherent ability of glutenin subunits to form disulfide bonds is thought to be determined by the primary and secondary structure of the proteins, which determines whether cysteine residues are present and available to form disulfide bonds, the capacity of a subunit to fold in the manner that would be required to form the bond, and the elasticity of the subunit once in the polymer to provide visco-elastic properties to a dough [2]. In this review, we discuss the structure of the glutenin macropolymer of wheat flour and dough.

Hydration of human nails investigated by NIR-FT-Raman spectroscopy

The human nail, although it is usually stable against outer influences, becomes soft and flexible after soaking in water. Frequent washing increases brittleness of nails. Hydration of nails is thought to be the most important factor influencing the physical properties of nails and possibly acts through changes in keratin structure. Here NIR-FT-Raman has been used to examine molecular structural changes of intact moisten nails. Raman spectra were obtained both in vitro from nail samples and in vivo before and after soaking in water. The water uptake of normal nail samples during the first 15 min was reflected in the increasing intensity ratio of the ν(OH)/ ν(CH 2) bands. A saturating effect appeared soon after 10 min which is explained by a defined water holding capacity. R( ν) representation of the low frequency range of the Raman spectra showed that mainly bound water is found both in dry and in wet nails. This implies water–protein interaction. Protein backbone vibration at 932 cm −1 indicating α-helical proteins increased in intensity in the wet nails. The ν(S–S) which is sensitive to changes in conformation of proteins showed a 4% higher intensity. Additional protein–water interactions could lead to a slight change of the dihedral angle of the C–S–S–C bonds and to geometric changes in coiling behavior of the α-helical protein. Suggesting a separation between matrix proteins and fiber proteins giving them a greater freedom of flexibility. The in vivo spectra detected from the distal part of the nail resembled spectra in vitro. Raman spectra of the proximal part of the nail showed that it was fully saturated with water. The proximal part of the nail did not show changes in water content and protein structure during nail moisturizing in the Raman spectra. Our results suggest that the softening of the nail following hydration may be due to changed matrix protein molecular structure induced by water.

Cysteine, glutathione (GSH) and zinc and copper ions together are effective, natural, intracellular inhibitors of (AIDS) viruses

Sufficient essential nutrients such as methionine, cysteine, copper, selenium, zinc and vitamins C and E are indispensable for the maintenance of optimal (immune) cell functions. Parasitic organisms such as protozoa, fungi, bacteria and viruses also depend on these essential nutrients for their multiplication and functioning. An evolutionarily developed optimal distribution of available nutrients between host (cells) and parasitic organisms normally prevents diseases, the nature of which will depend on genetic and environmental factors. The way in which the right amount of cysteine, glutathione (GSH), and copper and zinc ions made available in the right place at the right time and in the right form can prevent an unchecked multiplication of (AIDS) viruses in a more passive or active way forms the basis for the AIDS zinc-deficiency hypothesis (A–Z hypothesis) presented in this article.Zinc and copper ions stimulate/inhibit/block in a concentration-dependent way the (intracellular) activation of essential protein-splitting enzymes such as HIV proteases. Zinc and copper ions as ‘passive’ virus inhibitors. Apart from this, zinc ions directly or indirectly regulate, via zinc finger protein molecular structures, the activities of virus-combating Th-1 cells such as cytotoxic T-cells (CTLs). Zinc ions as regulators of the active, virus-combating Th-1 cells. Zinc and copper ions that remain available in sufficient amounts via cysteine/GSH are effective natural inhibitors/combaters of (AIDS) viruses and thereby prevent the development of chronic virus diseases that can lead to AIDS, autoimmune diseases, (food) allergies and/or cancer.A safe, relatively inexpensive and extensively tested medicine such as N-acetylcysteine (NAC) can help in supplying extra cysteine. The anti-HIV peptide T22, synthesized on the basis of two natural peptides from the Tachypleus tridentatus and Limnus polyphemus crabs, appears to be able to serve as supplier/carrier molecule of cysteine and zinc and/or to hinder the entry of HIVs into cells by way of the CD4 receptor.

Structures of the psychrophilic Alteromonas haloplanctis alpha-amylase give insights into cold adaptation at a molecular level.

. Enzymes from psychrophilic (cold-adapted) microorganisms operate at temperatures close to 0 degreesC, where the activity of their mesophilic and thermophilic counterparts is drastically reduced. It has generally been assumed that thermophily is associated with rigid proteins, whereas psychrophilic enzymes have a tendency to be more flexible. . Insights into the cold adaptation of proteins are gained on the basis of a psychrophilic protein's molecular structure. To this end, we have determined the structure of the recombinant form of a psychrophilic alpha-amylase from Alteromonas haloplanctis at 2.4 A resolution. We have compared this with the structure of the wild-type enzyme, recently solved at 2.0 A resolution, and with available structures of their mesophilic counterparts. These comparative studies have enabled us to identify possible determinants of cold adaptation. . We propose that an increased resilience of the molecular surface and a less rigid protein core, with less interdomain interactions, are determining factors of the conformational flexibility that allows efficient enzyme catalysis in cold environments.

Wilkins, Crick, and Watson: Nobel Prize for Work on the Structure of DNA

Maurice H. F. Wilkins, Francis H. C. Crick, and James D. Watson were recipients of the 1962 Nobel Prize in physiology or medicine for their work on the structure of deoxyribonucleic acid (DNA). Watson, who was profiled in a vignette published in the July 1996 issue of the Mayo Clinic Proceedings (page 658), was born in Chicago, Illinois, on Apr. 6,1928. He met Crick at the Cavendish Laboratory at Cambridge University (England) in 1951, where they collaborated on the Nobel Prizewinning work. Wilkins was born in Pongaroa, North Island, New Zealand, on Dec. 15, 1916. In 1938, he received a B.A. degree in physics from St. John's College in Cambridge and joined the Ministry of Home Security and Aircraft Production. He conducted graduate research in radar at the University of Birmingham and received a Ph.D. degree in 1940. His dissertation on luminescence and the movement of electrons in crystals contributed to improvements in radar screens. Subsequently, he worked on the separation of uranium isotopes for atomic weapons and was assigned to the University of California at Berkeley as a member of the Manhattan Project. In 1945, Wilkins became a lecturer in physics at St. Andrew's University in Scotland. The next year, he joined the staff of King's College at the University of London, where he studied the complex structure of the DNA molecule with ultrasonography and then with ultraviolet microscopy. He discovered a thin fiber of DNA, and with Rosalind E. Franklin (1920-1958), a colleague at King's College, he determined that the DNA molecule was shaped like a double helix, resembling a ladder twisted into a spiral. These findings were shared with Crick and Watson, who proposed the three-dimensional structure of the DNA molecule—a double helix consisting of two strands of sugar and phosphate joined by pairs of nitrogen bases within the helix. The helix bases are attached by hydrogen bonds, and when separated, a new molecule is synthesized by the opposite portion of each half of the original molecule. Wilkins was deputy director of the Medical Research Council Biophysics Unit at King's College from 1955 to 1970 and served as its director from 1970 to 1972. Subsequently, he became director of the Neurobiology Unit and headed the Cell Biophysics Unit from 1974 to 1980. He became professor emeritus in 1981. Crick was born in Northampton (northwest of London in central England) on Jun. 8,1916. In 1937, he received a B.S. degree in physics from University College in London. During World War II (1939-1945), he joined the British Admiralty Research Laboratory and participated in the development of mines. Influenced by What Is Life? The Physical Aspects of the Living Cell, a book written by Erwin Schrodinger (1887-1961), Crick became interested in biology, and in 1947, he began working at the Strangeways Research Laboratory in Cambridge. Subsequently, he worked on investigations into the molecular structure of proteins with Max F. Perutz (1914) at the Cavendish Laboratory at Cambridge University, where he met Watson in 1951. In 1953, Crickreceived his Ph.D. degree from Cambridge University for his work on the x-ray diffraction analysis of protein structure. He became director of the Molecular Biology Laboratory at Cambridge in 1962. In 1977, he joined the staff of the Salk Institute for Biological Studies in San Diego, California, where he conducted research on the mechanism of vision and the function of dreams. He also postulated that life on earth may have originated from microorganisms that were disseminated through space from another planet—the so-called panspermia hypothesis. Wilkins, Crick, and Watson were honored on a stamp issued by Sweden in 1989.

Mechanism of inducible nitric oxide synthase inactivation by aminoguanidine and L-N6-(1-iminoethyl)lysine.

The inducible nitric oxide synthase (iNOS) selective inhibitors aminoguanidine (AG) and N6-(1-iminoethyl)-L-lysine (NIL), under conditions that support catalytic turnover, inactivate the enzyme by altering in different ways the functionality of the active site. NIL inactivation of the iNOS primarily targets the heme residue at the active site, as evidenced by a time- and concentration-dependent loss of heme fluorescence that accompanies the loss of NO-forming activity. The NIL-inactivated iNOS dimers that have lost their heme partially disassemble into monomers with no fluorometrically detectable heme. AG inactivation of the iNOS is not accompanied by heme destruction, as evidenced by retention of heme fluorescence and absorbance after complete loss of NO-forming activity. The AG-inactivated iNOS dimers do not disassemble into monomers as extensively as NIL-inactivated dimers. Incubation of the iNOS with 14C-labeled NIL results in no detectable protein-associated radioactivity in the NIL-inactivated iNOS, suggesting that the primary mechanism of the iNOS inactivation by NIL is heme alteration and loss. In contrast, incubations of iNOS with 14C-labeled AG result in the incorporation of radioactivity into both iNOS protein and low molecular weight structures that migrate by SDS-PAGE similarly to free heme. These observations suggest that AG inactivation proceeds through multiple pathways of covalent modification of the iNOS protein and the heme residue at the active site, but which sustain the integrity of the heme porphyrin ring.

Introduction: electron microscopy for fungal cell ultrastructure

Ultrastructural morphology is a principal study for almost all natural science, i.e., an inevitable and fundamental study to elucidate structures, functions and differentiation of a given tissue, cell, or molecule as a most understandable visual form. The number of young scientists, however, who have been engaged in this field is very small recently, since this field appears to be too old when compared with modern molecular biology and it takes much longer time for the beginner to master the methology for electron microscopy (EM). This symposium is designed for these young scientists and molecular biologists or biochemists, who are not so familiar to ultrastructural morphology, to better understand the applicability of EM, through new results and findings in ultrastructures of fungal cells and related organisms. EM includes several kinds of methods, which are shadowing EM, negative staining EM, ultrathin section EM, scanning EM, freeze-fracture EM, immuno-EM and diverse methods for staining the specimen. Shadowing EM and negative staining EM are suitable methods for the study molecular structures of proteins, and the former is prepared by shadowing with platinum palladium in a vacuum chamber, and the latter is a method to observe a relief prepared by dipping the sample in phosphotungsten solution. Freeze-fracture electron microscopy is suitable for the study of membrane plane ultrastructures, since it reveals a wide planar view of the membrane by splitting it along the hydrophobic membrane internal plane. Immunoelectron microscopy is an essential method for the study of intracellular localization of proteinous molecules. These methods will be introduced. This symposium will introduce new findings as for fungal cells, bacteria and protozoa obtained principally by using electron microscopy. These findings obtained through ultrastructures may provided a renewed knowledge of research approach from view points of ultrastructure.

Functional analysis by imaging of melanophore pigment dispersion of chimeric receptors constructed by recombinant polymerase chain reaction

Analysis of functional aspects of the molecular structure of proteins often requires a means to selectively alter structure and subsequently analyze function. We have adapted a method of overlap extension polymerase chain reaction (PCR) to generate multiple domain replacements in G-protein coupled receptors. The examples described herein are β 2-adrenergic receptors whose G-protein coupling domains have been replaced by homologous domains of olfactory receptors, but the procedure has also been used to produce constructs with mutations, deletions, and fusions of two complete open reading frames. The chimeric olfactory-adrenergic receptors were assayed by functional expression in clonal lines of Xenopus melanophores. The ability of G-protein coupled second messenger pathways to cause translocation of pigment organelles within melanophores allows the use of video microscopy to assay the function of the chimeric receptors. Digital automation of microscope stage, camera, and image processing allows multiple parallel experiments to be performed. Melanophores allow responses mediated by the Gs, Gq and Gi pathways to be assayed with equal efficiency and the specificity of the coupling between chimera (or receptor) and G-protein subtypes can be rapidly determined.

Neglected aspects in the standardization of protein measurements. A standardized comparison method

Abstract

Two steps in the transition between the native and acid states of bovine α‐lactalbumin detected by circular polarization of luminescence: Evidence for a premolten globule state?

A few studies indirectly support the existence of an intermediate in the transition of Ca(2+)-saturated bovine alpha-lactalbumin (alpha-LA) from the native (N) to the acidic (A) state, known as the molten globule state. However, direct experimental evidence for the appearance of this intermediate has not been obtained. The signal of circular polarization of luminescence (CPL) is sensitive to fine conformational transitions because of its susceptibility to changes in the environmental asymmetry of fluorescent chromophores in their excited electronic states. In the present study, CPL measurements were applied using the intrinsic tryptophan fluorescence of alpha-LA as well as the fluorescence of 8-anilino-1-naphthalenesulfonic acid (ANS) bound to alpha-LA. CPL of tryptophan and ANS was measured in the pH range of 2.5-6 in order to find direct experimental evidence for the proposed intermediate. CPL (characterized by the emission anisotropy factor, g(em)) depends on the asymmetry of the protein molecular structure in the environment of the tryptophan and the ANS chromophores in the excited electronic state. The pH dependence of both the gab, absorption anisotropy factor determined by CD, and the ANS steady state fluorescence, showed a single transition at pH 3-3.7 as already reported elsewhere. This transition was interpreted as being a result of a change of the alpha-LA tertiary structure, which resulted in a loss of asymmetry of the environment of both the tryptophan residues and the ANS hydrophobic binding sites. The pH dependence of the tryptophan and ANS g(em) showed an additional conformational transition at pH 4-5, which coincided with the pKa of Ca2+ dissociation (pKa 5), as predicted by Permyakov et al. (1981, Biochem Biophys Res Commun 100:191-197). The titration curve showed that there is a pH range between 3.7 and 4.1 in which alpha-LA exists in an intermediate state between the N- and A-state. We suggest that the intermediate is the premolten globule state characterized by a reduced Ca2+ binding to the alpha-LA, native-like tertiary structure, and reduced asymmetric fluctuation of the tertiary structure on the nanosecond time scale. This intermediate resembles the "critical activated state" theoretically deduced by Kuwajima et al. (1989, J Mol Biol 206:547-561). The present study demonstrates the power of CPL measurements for the investigation of folding/unfolding transitions in proteins.

Discovery of new anticancer drugs by computer-aided drug design

Determination of the three-dimensional molecular structures of proteins, enzymes and nucleic acids is the first step in the development of molecular models of drug-receptor complexes that may be used in the rational computer-aided design of new and sometimes novel inhibitors with altered recognition and receptor-affinity properties. This approach provides a rational method for developing drugs that may be highly selective for a particular oncogene or other protein targets.