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Author: Elwy Hassan Abdelkader Ali Publisher: ISBN: Category : Languages : en Pages : 0
Book Description
Pulse electron paramagnetic resonance (EPR) distance measurements using double electron-electron resonance (DEER) experiments have been established as a powerful tool in structural biology. DEER experiments have the ability to measure the distance between two paramagnetic centres in biological macromolecules in the range of about 2 to 8 nm. The paramagnetic centres are usually introduced into proteins by site-directed spin labelling (SDSL) of cysteine residues. This thesis is based on the use of new lanthanide binding tags (LBTs) for paramagnetic nuclear magnetic resonance (NMR) spectroscopy (reported in papers 2 and 5), DEER distance measurements (reported in papers 1 and 3) and time-resolved luminescence resonance energy transfer (LRET) experiments (reported in paper 4). In particular, use of two complementary techniques, DEER experiments and paramagnetic NMR spectroscopy, was investigated for the study of conformational changes of proteins as a result of protein-ligand interactions. Two proteins were studied, the E. coli aspartate/glutamate binding protein (DEBP) and human calmodulin (CaM). Both proteins have different ligand binding characteristics: DEBP binds to small organic molecules, while CaM binds to specific peptide sequences. DEBP is a periplasmic binding protein responsible for the transport of aspartic acid and glutamic acid across the cell membrane and widely used in the design of biosensors of glutamate. The protein is composed of two domains, which bind one amino acid molecule at the domain interface. As DEBP contains a disulfide bond, an alternative cysteine-independent approach for site-specific protein tagging was used, which involved the use of genetically encoded unnatural amino acids that were site-specifically incorporated into proteins using orthogonal amber-suppressor tRNA/aminoacyl-tRNA synthetase systems. p-azido-L-phenylalanine (AzF) residues were incorporated into DEBP at different positions and paramagnetic lanthanide tags were attached to AzF via Cu(I)-catalyzed click chemistry (papers 1 and 2). Multiple Gd3+-Gd3+ distances measured by DEER experiments were used to define the metal positions, subsequently allowing deltachi-tensor determinations from sparse sets of pseudocontact shifts (PCSs). Both the DEER data and PCSs were in agreement with the closed conformation observed in the crystal structure of the homologue from S. flexneri. On the other hand, the PCSs indicated that the transition to the substrate-free protein involves a movement of the two domains as rigid entities relative to each other. CaM is a two-domain protein that acts as an intermediate messenger protein and intracellular calcium sensor, which responds to changes in Ca2+ concentrations by large conformational changes that enable binding to a range of different proteins involved in signalling pathways. The conformational changes of CaM upon binding of the myristoylated alanine-rich C-kinase substrate (MARCKS) peptide were studied using DEER experiments and paramagnetic NMR. MARCKS was chosen due to its unique binding mode compared to other CaM-target peptide complexes. The DEER results indicated that the binding of MARCKS peptide to CaM does not lock CaM in a single conformation. Deviations between the crystal and solution structure of the complex were also evident in the measured PCS data, highlighting the conformational flexibility of CaM that allows CaM to bind to diverse target proteins.
Author: Elwy Hassan Abdelkader Ali Publisher: ISBN: Category : Languages : en Pages : 0
Book Description
Pulse electron paramagnetic resonance (EPR) distance measurements using double electron-electron resonance (DEER) experiments have been established as a powerful tool in structural biology. DEER experiments have the ability to measure the distance between two paramagnetic centres in biological macromolecules in the range of about 2 to 8 nm. The paramagnetic centres are usually introduced into proteins by site-directed spin labelling (SDSL) of cysteine residues. This thesis is based on the use of new lanthanide binding tags (LBTs) for paramagnetic nuclear magnetic resonance (NMR) spectroscopy (reported in papers 2 and 5), DEER distance measurements (reported in papers 1 and 3) and time-resolved luminescence resonance energy transfer (LRET) experiments (reported in paper 4). In particular, use of two complementary techniques, DEER experiments and paramagnetic NMR spectroscopy, was investigated for the study of conformational changes of proteins as a result of protein-ligand interactions. Two proteins were studied, the E. coli aspartate/glutamate binding protein (DEBP) and human calmodulin (CaM). Both proteins have different ligand binding characteristics: DEBP binds to small organic molecules, while CaM binds to specific peptide sequences. DEBP is a periplasmic binding protein responsible for the transport of aspartic acid and glutamic acid across the cell membrane and widely used in the design of biosensors of glutamate. The protein is composed of two domains, which bind one amino acid molecule at the domain interface. As DEBP contains a disulfide bond, an alternative cysteine-independent approach for site-specific protein tagging was used, which involved the use of genetically encoded unnatural amino acids that were site-specifically incorporated into proteins using orthogonal amber-suppressor tRNA/aminoacyl-tRNA synthetase systems. p-azido-L-phenylalanine (AzF) residues were incorporated into DEBP at different positions and paramagnetic lanthanide tags were attached to AzF via Cu(I)-catalyzed click chemistry (papers 1 and 2). Multiple Gd3+-Gd3+ distances measured by DEER experiments were used to define the metal positions, subsequently allowing deltachi-tensor determinations from sparse sets of pseudocontact shifts (PCSs). Both the DEER data and PCSs were in agreement with the closed conformation observed in the crystal structure of the homologue from S. flexneri. On the other hand, the PCSs indicated that the transition to the substrate-free protein involves a movement of the two domains as rigid entities relative to each other. CaM is a two-domain protein that acts as an intermediate messenger protein and intracellular calcium sensor, which responds to changes in Ca2+ concentrations by large conformational changes that enable binding to a range of different proteins involved in signalling pathways. The conformational changes of CaM upon binding of the myristoylated alanine-rich C-kinase substrate (MARCKS) peptide were studied using DEER experiments and paramagnetic NMR. MARCKS was chosen due to its unique binding mode compared to other CaM-target peptide complexes. The DEER results indicated that the binding of MARCKS peptide to CaM does not lock CaM in a single conformation. Deviations between the crystal and solution structure of the complex were also evident in the measured PCS data, highlighting the conformational flexibility of CaM that allows CaM to bind to diverse target proteins.
Author: Stephen E. Harding Publisher: Oxford University Press, USA ISBN: 9780199637478 Category : Medical Languages : en Pages : 474
Book Description
This text on protein-ligand interactions offers a selection of the most useful and easily applied methods and acts as a guide to the principal techniques used.
Author: W. Guschlbauer Publisher: Springer ISBN: 9781468453843 Category : Science Languages : en Pages : 290
Book Description
This volume contains the texts of the nineteen lectures presented at the NATO-ASI - FEBS Course on "DNA - ligand interactions: from drugs to proteins." The Advanced Study Institute (ASIl was held from August 30th to September 11th. 1986 in the Abbey of Fontevraud (France). The ASI was attended by 112 participants from a wide scientific horizon and from twentyone different countries. It was in some way a follow-up of the ASI held in Maratea. Italy in May 1981 and which was published in the NATO ASI Life Science series as volume 45. While much has been learned about the way the cellular machinery maintains and transmits the genetic heritage. as well as how these processes are regulated. little is Known about how the interactions between the various partners involved are taKing place. The interactions of drugs and proteins with nucleic acids are of evident importance in the understanding of these problems. The spectacular advances in recombinant DNA technology and the increased sophistication of biophysical techniques. in particular >:-ray diffraction and nuclear magnetic resonance. have created a scientific environment which is highly promising for the future of research in molecular biology. These advances permH the serious hope that biology on the molecular level may become a r-eality. Some of the contributions at the ASI presented the most recent advances in this e>:citing field.
Author: Lawrence J. Berliner Publisher: Springer Science & Business Media ISBN: 1461528860 Category : Medical Languages : en Pages : 450
Book Description
The first of a two volume set, Volume 12 provides a long-awaited compilation of NMR theory to paramagnetic molecules. International experts report the latest developments in NMR methodology as applied to strongly relaxed and shifted resonances, detail the theoretical aspects of paramagnetic shift and relaxation, and discuss the interpretive bases of these molecular properties in relation to the structure and function of various paramagnetic molecules.
Author: Holger Gohlke Publisher: John Wiley & Sons ISBN: 3527645977 Category : Medical Languages : en Pages : 359
Book Description
Innovative and forward-looking, this volume focuses on recent achievements in this rapidly progressing field and looks at future potential for development. The first part provides a basic understanding of the factors governing protein-ligand interactions, followed by a comparison of key experimental methods (calorimetry, surface plasmon resonance, NMR) used in generating interaction data. The second half of the book is devoted to insilico methods of modeling and predicting molecular recognition and binding, ranging from first principles-based to approximate ones. Here, as elsewhere in the book, emphasis is placed on novel approaches and recent improvements to established methods. The final part looks at unresolved challenges, and the strategies to address them. With the content relevant for all drug classes and therapeutic fields, this is an inspiring and often-consulted guide to the complexity of protein-ligand interaction modeling and analysis for both novices and experts.
Author: Hans-Joachim Böhm Publisher: John Wiley & Sons ISBN: 3527605517 Category : Science Languages : en Pages : 262
Book Description
The lock-and-key principle formulated by Emil Fischer as early as the end of the 19th century has still not lost any of its significance for the life sciences. The basic aspects of ligand-protein interaction may be summarized under the term 'molecular recognition' and concern the specificity as well as stability of ligand binding. Molecular recognition is thus a central topic in the development of active substances, since stability and specificity determine whether a substance can be used as a drug. Nowadays, computer-aided prediction and intelligent molecular design make a large contribution to the constant search for, e. g., improved enzyme inhibitors, and new concepts such as that of pharmacophores are being developed. An up-to-date presentation of an eternally young topic, this book is an indispensable information source for chemists, biochemists and pharmacologists dealing with the binding of ligands to proteins.
Author: G. Ulrich Nienhaus Publisher: Humana ISBN: 9781617375255 Category : Science Languages : en Pages : 0
Book Description
A readily reproducible collection of established and emerging techniques for studying the interaction between proteins and ligands, including biochemical/bulk techniques, structure analysis, spectroscopy, single-molecule studies, and theoretical/computational tools. Among the highlights are surface plasmon resonance (SPR) and reflectometric biosensor approaches, high-throughput screening with confocal optics microscopy, single molecule fluorescence and fluorescence correlation spectroscopy (FCS), atomic force microscopy (AFM), crystallography of reaction intermediates, and time-resolved x-ray crystallography. The protocols follow the successful Methods in Molecular BiologyTM series format, each offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
Author: Yun Shi Publisher: ISBN: Category : Languages : en Pages : 207
Book Description
Protein-ligand interactions form the molecular basis of many biological processes. The study of their interactions from a structural perspective can provide not only insights into the molecular recognition between the protein and the ligand but also clues to the design of better ligands that can serve to mediate the biological events. This thesis investigates such interactions for four proteins that are (potential) therapeutic targets. Techniques used in this thesis include molecular dynamics (MD) simulations, saturation transfer difference (STD) NMR spectroscopy, and complete relaxation and conformational exchange matrix (CORCEMA) analysis that calculates theoretical STD effects. MD simulations are employed to study the binding of two designed glycopeptides with SYA/J6, a monoclonal antibody specific for the O-polysaccharide of the Shigella flexneri Y bacterium, as well as the binding dynamics and strengths of a series of inhibitors against human lactate dehydrogenase A (LDHA), an enzyme implicated in the cell energy metabolism of various cancers. The computational results from both cases are consistent with experimental data, predicting that neither glycopeptide would bind to SYA/J6, and clarifying ambiguities in the binding modes of two well-known LDHA inhibitors. Furthermore, binding models of two inhibitors against the enzyme UDP-galactopyranose mutase (UGM), a potential target for the treatment of tuberculosis, and two substrates of UDP-N-acetylgalactopyranose mutase (UNGM), a potential target against diarrheal disease, are constructed by a protocol that combines MD, STD NMR, and CORCEMA calculations. The collective results indicate a unique binding mode for a UGM inhibitor and explain the bifunctionality of UNGM.
Author: Siqi Guan Publisher: ISBN: Category : Languages : en Pages : 322
Book Description
Proteins are not static objects. They have a great variety of internal motions with different amplitudes and different timescales. These internal motions play an important role in catalytic processes. Therefore, the existence of an intimate relationship between protein dynamics and protein function is widely accepted. Due to the significance of protein dynamics, techniques have been developed to study protein dynamics including nuclear magnetic resonance (NMR) spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, and mass spectrometry (MS). Compared with NMR and EPR spectroscopy, MS has less stringent sample requirements, including protein concentration and protein size. Moreover, the mass accuracy, sensitivity, and faster data analysis also have contributed to the rapid growth of MS based techniques. Hydrogen-deuterium exchange mass spectrometry (HDX-MS), a combination of HPLC and MS, has become a common and sensitive tool to probe protein structural flexibility and solution dynamics. In this dissertation, HDX-MS was applied to study dynamic changes of proteins due to substrate binding and protein-protein interactions. The GT-A glycosyltransferase glucosyl-3-phosphoglycerate synthase from Mycobacterium tuberculosis (MtGpgS) catalyzes the first step of biosynthesis of 6-O-methylglucose lipopolysaccharides (MGLPs), which are essential to growth and existence of mycobacterium. The HDX-MS data revealed that the two substrates UDP-glucose (UDPG) and 3-phosphoglycerate (3PGA) can bind to MtGpgS independently, disagreeing with the previous proposal that 3PGA can only bind to MtGpgS after UDPG. Moreover, 3PGA was found to bind to or allosterically affect the UDPG binding site. Furthermore, the HDX-MS data revealed that MtGpgS may provide a necessary conformation for UDPG binding, while it goes through a large conformational change on 3PGA binding. The GT-B glycosyltransferase MshA from Corynebacterium glutamicum (CgMshA) catalyzes the initial step of mycothiol biosynthesis. A large conformational change was observed in CgMshA on nucleotide binding by superimposing APO structure of CgMshA and complex structure with UDP. HDX-MS was utilized to study conformational changes of CgMshA on substrate binding from the aspect of dynamics, providing a complementary to static structures. The HDX-MS data showed that both substrates uridine diphosphate glucose-N-acetylglucosamine (UDP-GlcNAc) and 1-L-myo-inositol-1-phosphate (I1P) can bind to CgMshA independently, but the I1P binding is not productive since it binds to an uncorrect site. Moreover, the I1P binding can lead to dynamic changes of CgMshA, while only UDP-GlcNAc can induce the major conformational change of CgMshA. Furthermore, the 3PGA binding cannot induce further dynamic changes of CgMshA in the presence of UDP. HDX-MS was also employed to study dynamic changes of protein complex SufBC2D from Escherichia coli on ADP/Mg2+ binding. This complex is responsible for Fe-S cluster assembly under oxidative stress. The crystal structure of SufBC2D complex has been determined, while little dynamic information is known. So HDX-MS was applied to study dynamic changes of the SufBC2D complex. The HDX-MS data revealed that SufC has a significant conformational change, which may be required by ATP binding and hydrolysis. Moreover, SufB and SufD are detected to have dynamic changes due to SufC conformational changes. These dynamic changes suggest that SufB-SufD protomer may have a conformational change in order to provide a suitable conformation for Fe-S cluster assembly. This work demonstrates that HDX-MS can be effectively used to study protein-ligand and protein-protein interactions, as well as the accompanying changes in structural dynamics. HDX-MS data detects substrate binding mechanism and conformational changes that are not available through x-ray crystallography. With these advantages, HDX-MS has been applied in study of protein structure and dynamics, studying protein-ligand and protein-protein interactions, protein folding, as well as protein therapeutics discovery and development.
Author: Elwy Hassan Abdelkader Ali
Author: Elwy Hassan Abdelkader Ali
Author: Stephen E. Harding
Author: W. Guschlbauer
Author: Christopher R. McCullough
Author: Lawrence J. Berliner
Author: Holger Gohlke
Author: Hans-Joachim Böhm
Author: G. Ulrich Nienhaus
Author: Yun Shi
Author: Siqi Guan