Characterization of enzyme motions by solution NMR relaxation dispersion

In many enzymes, conformational changes that occur along the reaction coordinate can pose a bottleneck to the rate of conversion of substrates to products. Characterization of these rate-limiting protein motions is essential for obtaining a full understanding of enzyme-catalyzed reactions. Solution NMR experiments such as the Carr-Purcell-Meiboom-Gill (CPMG) spin-echo or off-resonance R 1rho pulse sequences enable quantitation of protein motions in the time range of microseconds to milliseconds. These experiments allow characterization of the conformational exchange rate constant, k ex, the equilibrium populations of the relevant conformations, and the chemical shift differences (Deltaomega) between the conformations. The CPMG experiments were applied to the backbone N-H positions of ribonuclease A (RNase A). To probe the role of dynamic processes in the catalytic cycle of RNase A, stable mimics of the apo enzyme (E), enzyme-substrate (ES) complex, and enzyme-product (EP) complex were formed. The results indicate that the ligand has relatively little influence on the kinetics of motion, which occurs at 1700 s (-1) and is the same as both k cat, and the product dissociation rate constant. Instead, the effect of ligand is to stabilize one of the pre-existing conformations. Thus, these NMR experiments indicate that the conformational change in RNase A is ligand-stabilized and does not appear to be ligand-induced. Further evidence for the coupling of motion and enzyme function comes from the similar solvent deuterium kinetic isotope effect on k ex derived from the NMR measurements and k cat from enzyme kinetic studies. This isotope effect of approximately 2 depends linearly on solvent deuterium content suggesting the involvement of a single proton in RNase A motion and function. Moreover, mutation of His48 to alanine eliminates motion in RNase A and decreases the catalytic turnover rate indicating the involvement of His48, which is far from the active site, in coupling motion and function. For the enzyme triosephosphate isomerase (TIM), the opening and closing motion of a highly conserved active site loop (loop 6) has been implicated in many studies to play an important role in the catalytic cycle of the enzyme. Off-resonance R 1rho experiments were performed on TIM, and results were obtained for amino acid residues in the N-terminal (Val167), and C-terminal (Lys174, Thr177) portions of loop 6. The results indicate that all three loop residues move between the open and closed conformation at about 10,000 s (-1), which is the same as the catalytic rate constant. The O (eta) atom of Tyr208 provides a hydrogen bond to stabilize the closed form of loop 6 by interacting with the amide nitrogen of Ala176; these atoms are outside of hydrogen bonding distance in the open form of the enzyme. Mutation of Tyr208 to phenylalanine results in significant loss of catalytic activity but does not appear to alter the kex value of the N-terminal part of loop 6. Instead, removal of this hydrogen bond appears to result in an increase in the equilibrium population of the open conformer of loop 6, thereby resulting in a loss of activity through a shift in the conformational equilibrium of loop 6. Solution NMR relaxation dispersion experiments are powerful experimental tools that can elucidate protein motions with atomic resolution and can provide insight into the role of these motions in biological function.     

Hydration of amino acid side chains: dependence on secondary structure

Energy calculations have been used to study the hydration sitesaround the polar groups of serine, threonine and tyrosine sidechains. These hydration sites depend not only on the hybridizationof the polar group but also on the local secondary structure,the X₁ side chain torsion angle and the position of the hydroxylhydrogen atom. For tyrosine side chains, two solvent sites arefound approximately in the plane of the ring. Even for serineand threonine side chains only two minimum energy sites arefound in general of which one is in an expected position withinhydrogen bonding of the hydroxyl hydrogen atom (unless thisis blocked from interaction with solvent molecules by, for example,O_i–4 or O_i–3. The position of the second of thesesites depends not only on the position of the hydroxyl oxygenbut also on neighbouring main chain atoms to which it can alsohydrogen bond. There is good agreement with the solvent distributionsobtained from crystallographic data.     

Molecular dynamics and NMR spin relaxation in proteins

Molecular dynamics simulations often play a central role in the analysis of biomolecular NMR data. The focus here is on NMR spin-relaxation, which can provide unique insights into the time-dependence of conformational fluctuations, especially on picosecond to nanosecond time scales which can be directly probed by simulations. A great deal has been learned from such simulations about the general nature of such motions and their impact on NMR observables. In principle, relaxation measurements should also provide valuable benchmarks for judging the quantitative accuracy of simulations, but there are a variety of experimental and computational obstacles to making useful direct comparisons. It seems likely that simulations on time scales that are just now becoming generally feasible may provide important new information on internal motions, overall rotational diffusion, and the coupling between internal and rotational motion. Such information could provide a sound foundation for a new generation of detailed interpretation of NMR spin-relaxation results.     

Characterization of the conformational changes of partially hydrolyzed polyacrylamide chains in solution using stress recovery experiment

In this study, a simple empirical decay function model is fitted to the experimental data to characterize the conformational changes of partially hydrolyzed polyacrylamide (HPAM) in semidilute solutions, using the stress recovery experiment after cessation of a steady shear flow processing. With the increase of HPAM concentration and NaCl concentration, the parameter κ decreases, and this is in excellent agreement with the conformational changes of HPAM molecule chains. Experimental results show that the parameter κ can provide some information on the changes of the polymer chain conformation. the polymer chain conformation. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 2709–2713, 2006     

pH responsive polymers with amino acids in the side chains and their potential applications

Design and synthesis of pH responsive polymeric materials has become an important subject in academia as well as in industrial field in recent years due to their applications in diverse field including controlled drug delivery, biomedical applications, membrane science, sensors and actuators, oil recovery, colloid stabilization, etc. Efforts have been made to incorporate stimuli‐responsive biomolecules in synthetic polymers to develop pH responsive “smart” non‐biological hybrid macromolecules with high water solubility, enhanced biocompatibility, bio‐mimetic structure and properties. This review is focused on the recent advances in side‐chain amino acid‐based pH responsive polymers synthesis and potential application aspects of these macromolecular architectures in drug and gene delivery, and other fields. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 41084.     

Fluorinated polyacrylates containing amino side chains for the surface modification of waterborne epoxy resin

Fluorinated polyacrylates containing amino side chains (FACAs) were synthesized to improve the water repellency of waterborne epoxy resin. First, FACA was prepared by a two-step process. Then, a phase-inversion emulsion technique, taking FACA as the reactive surface additives, was employed to prepare waterborne epoxy resin. The effect of the fluorinated content and the length of the fluorine side chain on the surface, thermal, and bulk properties of the waterborne epoxy resin were investigated. It was observed that moderate increase of the fluorinated content or the length of the fluorine side chain improved the contact angle and thermal properties while the mechanical property was not deteriorated. A small amount of fluoride (0.07 wt %) in the EA-F1/3-1 sample can dramatically increase the water contact angle from 72.63° to 95.31° due to the strong tendency of the macromonomers to migrate toward the outmost layer, resulting in enrichment of fluorine atoms on the surface. X-ray photoelectron spectroscopy results revealed that for a given weight of the two macromonomers, F atomic concentration of the copolymer modified by longer fluorinated side chains was higher than that modified by short fluorinated side chains. From the present investigation, FACA successfully improved the surface property of waterborne epoxy resin and showed a prominent potential application in large-scale industrialization. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47091.     

Structural mobility of molecular bottle-brushes investigated by NMR relaxation dynamics

The structural mobility of monomeric units of molecular bottle-brushes was studied by a systematic evaluation of NMR relaxation dynamics. The spin-spin relaxation time (T₂) was determined by Carr-Purcell-Meiboom-Gill (CPMG) NMR spectroscopic measurements. T₂ for protons that reside on the exterior and interior of the bottle-brush macromolecules varied with the grafting density and side chain length in bottle-brush copolymers. Poly((2-(2-bromopropionyloxy)ethyl methacrylate-stat-methyl methacrylate)-graft-butyl acrylate) (poly((BPEM-stat-MMA)-graft-PBA) was studied as a model brush copolymer. The T₂ values for protons of MMA units in the brush backbone significantly decreased with increasing side chain length and grafting density of PBA. The mobility and relaxation times T₂ for the side chain PBA protons decreased with grafting density. However, after initial increase, the relaxation times eventually decreased with PBA side chain length.     

Characterization of siloxane copolymers by solution O NMR spectroscopy

Solution ¹⁷O NMR spectroscopy was used for structure elucidation of siloxane copolymers with the natural abundance of ¹⁷O, i.e. without any enrichment prior to spectroscopy. Homo, co, and terpolymers, as well as linear chains, cyclic oligomers, and graft polymers were investigated. All relevant chemical shifts and corresponding linewidths were reported for siloxane polymers substituted with methyl, phenyl, 3-cyanopropyl, 2-cyanoethyl, 3,3,3-trifluoropropyl, and polyethylene glycol ligands and with the backbone stiffening groups tetramethyl-p-silphenylene, tetramethyl-p,p′-sildiphenylene ether, and m-carborane. An increment system was extended to predict the chemical shifts of substituted siloxane copolymers. ¹⁷O NMR spectroscopy of polysiloxanes provided information concerning their chemical composition, average molecular weight, and microstructure.     

Efficiently removing phenols from aqueous solution using amino acid functionalized D301 resins

ABSTRACT

Amino acid functionalized D301 resins (Tyr-D301, Phe-D301, and Ala-D301) are successfully prepared to remove phenols. The influences of adsorption condition on adsorption ability and the practical application value are investigated. The functionalized D301 resins possess strong adsorption ability toward phenol by ways of hydrogen bond interaction. The adsorption capacity of Tyr-D301, Phe-D301, and Ala-D301 is 388.2, 368.4, and 321.5 mg· g^?1 at 293 K and pH of 2, respectively. The adsorption process could be described satisfactorily by Lagergren-first-order model and Langmuir model. The rate constant is 0.057, 0.042, and 0.035 min^?1, respectively. The functionalized D301 can be used to dispose wastewater containing phenols.     

Determination of the amino acid tryptophan in protein fibres

Tryptophan can be used as an indicator amino acid for the photostability of proteins. The analysis of tryptophan and its degradation products is hampered by their instability in oxidative or strongly acidic media, or light. Various methods were employed to quantify tryptophan in wool and other protein fibres such as silk and human hair. Acid, alkaline and enzymatic methods were used to hydrolyse protein fibres. The amino acid tryptophan in wool and other protein fibres was determined by a colorimetric method, by amino acid analysis and by reversed-phase HPLC. The different analytical methods were compared with regard to their results. The colorimetric method (p-dimethylamino-benzaldehyde) proved to give reliable results for the tryptophan content in wool and unpigmented protein fibres. This method can be used also for wool dyed with acid, metal complex or reactive dyes or for pigmented keratin fibres after correction for a blank sample. Interference between dye and colorimetric reagent also have to be evaluated.     

氨基酸扩散系数的分子动力学模拟

采用分子动力学模拟的方法模拟了298. 15 K正则系综下甘氨酸、丙氨酸等12种氨基酸在水中的扩散过程，扩散系数的计算采用微分-区限变分法。计算结果表明：相同浓度下按不同分子数样本计算得到的扩散系数有较大差别，且分子数越多则模拟结果越接近于实验值。5种氨基酸在水中的扩散系数与文献值相对比，误差小于7%。还用同样的方法模拟了氧气在水中的扩散过程，模拟结果与实验结果吻合也较好。实验表明采用分子模拟手段可以获得具有工业应用价值的扩散系数，从而有助于计算传质学的发展。     

Molecular dynamics simulation of winter flounder antifreeze protein variants in solution: correlation between side chain spacing and ice lattice

The solution structure of the 38 amino acid C-terminal regionof the precursor for the HPLC-6 antifreeze protein from winterflounder has been investigated with molecular dynamics usingthe AMBER software. The simulation for the peptide in aqueoussolution was carried out at a constant temperature of 0°Cand at atmospheric pressure. The simulation covered 120 ps andthe results were analyzed based on data sampled upon reachinga stable equilibrium phase. Information has been obtained onthe quality of constant temperature and pressure simulations,the solution structure and dynamics, the hydrogen bonding network,the helix-stabilizing role of terminal charges and the interactionwith the surrounding water molecules. The Lys18–Glu22interactions and the terminal charged residues are found tostabilize a helical structure with the side chains of Thr2,Thr13, Thr24 and Thr35 equally spaced on one side of the helix.The spacing between oxygen atoms in the hydroxyl group of thethreonine side chains exhibits fluctuations of the order of2–3 Å during the 120 ps of simulation, but valuessimultaneously close to the repeat distance of 16.6 Åbetween oxygen atoms along the [0112] direction in ice are observed.Furthermore, two engineered variants were studied using thesame simulation protocol.     

Dynamics of macromolecular chains. 13C spin relaxation study of short-chain polystyrenes in deutero-chloroform solution

The results of T₁, T₂ and nuclear Overhauser effect (NOE) measurements on molecular weights 510–110 000 at weight fractions 0.05–0.60 in the title system suggest that previously neglected entanglement effects are highly significant for the reorientational processes in polymer chains and that previous T₂ estimates from bandwidths may be too low. T₂ is usually equal to T₁, and deviates only at high concentrations in these systems. The data can be represented by a double exponential reorientational correlation function of the form

$\text{G(τ)= A}\text{exp}\text{(}\text{? τ}\text{τ}{}_{\mathrm{A}}\text{) + B(}\text{? τ}\text{τ}{}_{\mathrm{B}}\text{)}$

where A + B = 1. The correlation times τ_A and τ_B are typically 1.5 × 10^?10 and 1.5 × 10^?9s, respectively. B increases with increasing concentration and molecular weight. The terminal phenyl group rotation is quite free in contrast to the backbone phenyls, and a spinning ratio above 15 has been estimated in a 60% solution for this group. Signal assignments and relaxation times (T₁) are given for 15 different ¹³C signals of these polymers at high concentrations.     

P(St-MMA)聚合物在CDCl_3溶剂中的核磁共振弛豫及高分子链运动

采用1H,13C核磁共振技术,分别指认了P(St-MMA)聚合物氢谱、碳谱的各个信号,并通过对P(St-MMA)共聚物在CDCl3溶剂中变温13C-NMR和1H-NMR弛豫时间的测量,证实了溶液中甲基丙烯酸甲酯—苯乙烯共聚物体系中高分子链间的凝聚缠结结构的存在,并分析了缠结对温度的依赖性。     

NMR relaxation studies of the role of conformational entropy in protein stability and ligand binding

Recent advances in the measurement and analysis of protein NMR relaxation data have made it possible to characterize the dynamical properties of many backbone and side chain groups. With certain caveats, changes in flexibility that occur upon ligand binding, mutation, or changes in sample conditions can be interpreted in terms of contributions to conformational entropy. Backbone and side chain flexibility can either decrease or increase upon ligand binding. Decreases are often associated with "enthalpy-entropy compensation" and "induced fit" binding, whereas increases in conformational entropy can contribute to stabilization of complexes. In certain cases, conformational entropy appears to play a role in cooperative binding and enzyme catalysis. In addition, variations in conformational entropy and heat capacity may both be important in stabilizing the folded structures of proteins.