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Amide Protons (amide + proton)
Selected AbstractsHydrogen-deuterium exchange in membrane proteins monitored by IR spectroscopy: A new tool to resolve protein structure and dynamicsBIOPOLYMERS, Issue 1-2 2004C. Vigano Abstract As more and more high-resolution structures of proteins become available, the new challenge is the understanding of these small conformational changes that are responsible for protein activity. Specialized difference Fourier transform infrared (FTIR) techniques allow the recording of side-chain modifications or minute secondary structure changes. Yet, large domain movements remain usually unnoticed. FTIR spectroscopy provides a unique opportunity to record 1H/2H exchange kinetics at the level of the amide proton. This approach is extremely sensitive to tertiary structure changes and yields quantitative data on domain/domain interactions. An experimental setup designed for attenuated total reflection and a specific approach for the analysis of the results is described. The study of one membrane protein, the gastric H+,K+ -ATPase, demonstrates the usefulness of 1H/2H exchange kinetics for the understanding of the molecular movement related to the catalytic activity. © 2004 Wiley Periodicals, Inc. Biopolymers, 2004 [source] PDK1 and PKB/Akt: Ideal Targets for Development of New Strategies to Structure-Based Drug DesignIUBMB LIFE, Issue 3 2003Thomas Harris Abstract Growth factor binding events to receptor tyrosine kinases result in activation of phosphatidylinositol 3-kinase (PI3K), and activated PI3K generates the membrane-bound second messengers phosphatidylinositol 3,4-diphosphate [PI(3,4)P2] and PI(3,4,5)P3, which mediate membrane translocation of the phosphoinositide-dependent kinase-1 (PDK1) and protein kinase B (PKB, also known as Akt). In addition to the kinase domain, PDK1 and PKB contain a pleckstrin homology (PH) domain that binds to the second messenger, resulting in the phosphorylation and activation of PKB by PDK1. Recent evidence indicates that constitutive activation of PKB contributes to cancer progression by promoting proliferation and increased cell survival. The indicating of PDK1 and PKB as primary targets for discovery of anticancer drugs, together with the observations that both PDK1 and PKB contain small-molecule regulatory binding sites that may be in proximity to the kinase active site, make PDK1 and PKB ideal targets for the development of new strategies to structure-based drug design. While X-ray structures have been reported for the kinase domains of PDK1 and PKB, no suitable crystals have been obtained for either PDK1 or PKB with their PH domains intact. In this regard, a novel structure-based strategy is proposed, which utilizes segmental isotopic labeling of the PH domain in combination with site-directed spin labeling of the kinase active site. Then, long-range distance restraints between the 15N-labeled backbone amide groups of the PH domain and the unpaired electron of the active site spin label can be determined from magnetic resonance studies of the enhancement effect that the paramagnetic spin label has on the nuclear relaxation rates of the amide protons. The determination of the structure and position of the PH domain with respect to the known X-ray structure of the kinase active site could be useful in the rational design of potent and selective inhibitors of PDK1 and PKB by 'linking' the free energies of binding of substrate (ATP) analogs with analogs of the inositol polar head group of the phospholipid second messenger. The combined use of X-ray crystallography, segmental isotopic and spin labeling, and magnetic resonance studies can be further extended to the study of other dynamic multidomain proteins and targets for structure-based drug design. IUBMB Life, 55: 117-126, 2003 [source] Temperature dependencies of amide 1H- and 15N-chemical shifts in hyaluronan oligosaccharidesMAGNETIC RESONANCE IN CHEMISTRY, Issue 5 2007Charles D. Blundell Abstract Temperature coefficients (,,/,T) of amide chemical shifts of N -acetylglucosamine residues have been measured in a range of oligosaccharides of the important vertebrate polysaccharide hyaluronan. Odd- and even-numbered oligosaccharides with glucuronic acid, ,-4,5-unsaturated glucuronic acid and N -acetylglucosamine at the termini were investigated. All amide proton temperature coefficients were only slightly less negative (,6.9 to , 9.1 ppb/ °C) than those of amide protons in free exchange with water (,, 11 ppb/ °C), indicating an absence of persistent intramolecular hydrogen bonds. With the exception of amide groups in reducing-terminal N -acetylglucosamine rings, all amide proton environments have the same temperature coefficient (,6.9 ppb/ °C), irrespective of differences in amide group chemical shifts and 3JHH coupling constants, i.e. they do not sense subtle differences in orientation of the amide group. Amide nitrogen temperature coefficients report the same phenomena but with greater sensitivity. These data provide a set of reference values for temperature coefficients measured in other carbohydrates with acetamido sugars. Copyright © 2007 John Wiley & Sons, Ltd. [source] Identification of bound waters in the solution structure of ribonuclease T1 using the double pulsed field gradient spin-echo NMR technique for selective water excitationMAGNETIC RESONANCE IN CHEMISTRY, Issue 9 2002Mitsuru Tashiro Abstract Novel pulse sequences incorporating the double pulsed field gradient spin-echo technique are presented that have particular use in identifying macromolecular bound water. The use of these sequences is illustrated using ribonuclease T1. Five amide protons cross-relaxing with bound water protons were observed. Examination of the crystal structure revealed that all of these amide protons donate hydrogen bonds or are in close proximity to water molecules with very low temperature factors, indicating that these amide protons are highly correlated with the bound water molecules. This method rapidly provides reliable information for characterizing macromolecular bound water molecules. Copyright © 2002 John Wiley & Sons, Ltd. [source] Amide proton hydrogen exchange rates for sperm whale myoglobin obtained from 15N- 1H NMR spectraPROTEIN SCIENCE, Issue 1 2000Silvia Cavagnero Abstract The hydrogen exchange behavior of exchangeable protons in proteins can provide important information for understanding the principles of protein structure and function. The positions and exchange rates of the slowly-exchanging amide protons in sperm whale myoglobin have been mapped using 15N- 1H NMR spectroscopy. The slowest-exchanging amide protons are those that are hydrogen bonded in the longest helices, including members of the B, E, and H helices. Significant protection factors were observed also in the A, C, and G helices, and for a few residues in the D and F helices. Knowledge of the identity of slowly-exchanging amide protons forms the basis for the extensive quench-flow kinetic folding experiments that have been performed for myoglobin, and gives insights into the tertiary interactions and dynamics in the protein. [source] Isomeric Squaraine-Based [2]Pseudorotaxanes and [2]Rotaxanes: Synthesis, Optical Properties, and Their Tubular Structures in the Solid StateCHEMISTRY - A EUROPEAN JOURNAL, Issue 28 2010Min Xue Abstract On the basis of formation of [2]pseudorotaxane complexes between triptycene-derived tetralactam macrocycles 1,a and 1,b and squaraine dyes, construction of squaraine-based [2]rotaxanes through clipping reactions were studied in detail. As a result, when two symmetrical squaraines 2,d and 2,e were utilized as templates, two pairs of isomeric [2]rotaxanes 3,a,b and 4,a,b as diastereomers were obtained, owing to the two possible linking modes of triptycene derivatives. It was also found, interestingly, that when a nonsymmetrical dye 2,g was involved, there existed simultaneously three isomers of [2]rotaxanes in one reaction due to the different directions of the guest threading. The 1H,NMR and 2D NOESY NMR spectra were used to distinguish the isomers, and the yield of [2]rotaxane 5,a with the benzyl group in the wider rim of the host 1,a was found to be higher than that of another isomer 5,b with an opposite direction of the guest, which indicated the partial selection of the threading direction. The X-ray structures of 3,b and 4,a showed that, except for the standard hydrogen bonds between the amide protons of the hosts and the carbonyl oxygen atoms of the guests, multiple ,,,,, stacking and CH,,,, interactions between triptycene subunits and aromatic rings of the guests also participated in the complexation. Crystallographic studies also revealed that the [2]rotaxane molecules 3,b and 4,a further self-assembled into tubular structures in the solid state with the squaraine dyes inside the channels. In the case of 4,a, all the nonsymmetrical macrocyclic molecules pointed in one direction, which suggests the formation of oriented tubular structures. Moreover, it was also found that the squaraines encapsulated in the triptycene-derived macrocycles were protected from chemical attack, and subsequently have potential applications in imaging probes and other biomedical areas. [source] Study of Structural Stability of Cyclophilin A by NMR and Circular Dichroism SpectraCHINESE JOURNAL OF CHEMISTRY, Issue 7 2006Yan-Hong Shi Abstract The structural stability of cyclophilin A (CypA) was investigated using H/D exchange and temperature coefficients of chemical shifts of amide protons, monitored by 2D heteronuclear NMR spectroscopy. Amide proton exchange rates were measured by H/D exchange experiments for slow-exchange protons and measured by SEA (Solvent Exposed Amides)-HSQC experiments for fast-exchange protons. Temperature coefficients of chemical shifts and hydrogen exchange rates of amide protons show reasonably good correlation with the protein structure. Totally, 44 out of 153 non-proline assigned residues still exist in 86 d of hydrogen-deuterium exchange at 4 °C, suggesting that CypA structure should be highly stable. Residues in secondary structures of ,2, ,1, ,2, ,5, ,6 and ,7 might constitute the hydrophobic core of the protein. The change in free energy of unfolding (,Gu) of CypA was estimated to be (21.99±1.53) kJ·mol,1 by circular dichroism (CD). The large free energy change is also an indicator of the high structural stability. [source] Clean SEA-TROW experiments to map solvent exposed amides in large proteinsCHINESE JOURNAL OF CHEMISTRY, Issue 12 2004Dong-Hai Lin Abstract It is well known that the SEA-TROSY experiment could alleviate some of the problems of resonance overlap in 15N/2H labeled proteins as it was designed to selectively map solvent exposed amide protons. However, SEA-TROSY spectra may be contaminated with exchange-relayed NOE contributions from fast exchanged hydroxyl or amine protons and contributions from longitudinal relaxation. Also, perdeuteration of the protein sample is a prerequisite for this experiment. In this communication, a modified version, clean SEA-TROSY, was proposed to eliminate these artifacts and to allow the experiment to be applied to protonated or partially deuterated proteins and protein complexes. [source] | ||||||||||