Lishan Yao

NMR Determination of Amide N-H Equilibrium Bond Length from Concerted Dipolar Coupling Measurements

The N−H bond length in backbone peptide groups of the protein GB3 has been studied by liquid-crystal NMR, using five structurally conserved mutants of this protein. In the absence of additional information, the impact of dynamic fluctuations of the N−H vector orientation on the 15N−1H dipolar interaction cannot be separated from a change in N−H bond length. However, a change in N−H bond length directly impacts the orientation of C′−H vectors in the peptide group, and simultaneous analysis of 13C′−HN and 15N−HN residual dipolar couplings, measured under five different alignment orientations, permits modelfree determination of the average equilibrium N−H bond length in GB3, yielding rNHeq = 1.008 ± 0.006 A. Anharmonicity of the bond stretching results in a slightly longer time-averaged bond length = 1.015 ± 0.006 A, and an effective bond length reff = −1/3 = 1.023 ± 0.006 A pertinent for NMR relaxation analysis, not including the impact of zero-point or other angular fluctuations in N−H orienta...

The Impact of Hydrogen Bonding on Amide 1H Chemical Shift Anisotropy Studied by Cross-Correlated Relaxation and Liquid Crystal NMR Spectroscopy

Site-specific 1H chemical shift anisotropy (CSA) tensors have been derived for the well-ordered backbone amide moieties in the B3 domain of protein G (GB3). Experimental input data include residual chemical shift anisotropy (RCSA), measured in six mutants that align differently relative to the static magnetic field when dissolved in a liquid crystalline Pf1 suspension, and cross-correlated relaxation rates between the 1HN CSA tensor and either the 1H−15N, the 1H−13C′, or the 1H−13Cα dipolar interactions. Analyses with the assumption that the 1HN CSA tensor is symmetric with respect to the peptide plane (three-parameter fit) or without this premise (five-parameter fit) yield very similar results, confirming the robustness of the experimental input data, and that, to a good approximation, one of the principal components orients orthogonal to the peptide plane. 1HN CSA tensors are found to deviate strongly from axial symmetry, with the most shielded tensor component roughly parallel to the N−H vector, and the least shielded component orthogonal to the peptide plane. DFT calculations on pairs of N-methyl acetamide and acetamide in H-bonded geometries taken from the GB3 X-ray structure correlate with experimental data and indicate that H-bonding effects dominate variations in the 1HN CSA. Using experimentally derived 1HN CSA tensors, the optimal relaxation interference effect needed for narrowest 1HN TROSY line widths is found at ∼1200 MHz.

Liquid crystalline phase of G-tetrad DNA for NMR study of detergent-solubilized proteins.

The liquid crystalline phase consisting of the potassium salt of the dinucleotide d(GpG) is compatible with detergents commonly used for solubilizing membrane proteins, including dodecylphosphocholine, the lysolipid 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine, and small bicelles consisting of dihexanoyl phosphatidylcholine and dimyristoyl phosphatidylcholine. The chiral nematic liquid crystalline phase of d(GpG) consists of long columns of stacked G-tetrad structures and carry a net negative charge. For water-soluble systems, the protein alignment induced by d(GpG) is very similar to that observed for liquid crystalline Pf1 bacteriophage, but of opposite sign. Alignment of the detergent-solubilized fusion domain of hemagglutinin is demonstrated to be homogeneous and stable, resulting in high quality NMR spectra suitable for the measurement of residual dipolar couplings.

Correlated dynamics between protein HN and HC bonds observed by NMR cross relaxation

Although collective dynamics of atom groups steer many biologically relevant processes in biomacromolecules, most atomic resolution motional studies focus on isolated bonds. In this study, a new method is introduced to assess correlated dynamics between bond vectors by cross relaxation nuclear magnetic resonance (NMR). Dipole-dipole cross correlated relaxation rates between intra- and inter-residual H(N)-N and H(alpha)-C(alpha) in the 56 residue protein GB3 are measured with high accuracy. It is demonstrated that the assumption of anisotropic molecular tumbling is necessary to evaluate rates accurately and predictions from the static structure using effective bond lengths of 1.041 and 1.117 A for H(N)-N and H(alpha)-C(alpha) are within 3% of both experimental intra- and inter-residual rates. Deviations are matched to models of different degrees of motional correlation. These models are based on previously determined orientations and motional amplitudes from residual dipolar couplings with high accuracy and precision. Clear evidence of correlated motion in the loops comprising residues 10-14, 20-22, and 47-50 and anticorrelated motion in the alpha helix comprising 23-38 is presented. Somewhat weaker correlation is observed in the beta strands 2-4, which have previously been shown to exhibit slow correlated motional modes.

New Reactions and Products Resulting from Alternative Interactions between the P450 Enzyme and Redox Partners.

Cytochrome P450 enzymes are capable of catalyzing a great variety of synthetically useful reactions such as selective C–H functionalization. Surrogate redox partners are widely used for reconstitution of P450 activity based on the assumption that the choice of these auxiliary proteins or their mode of action does not affect the type and selectivity of reactions catalyzed by P450s. Herein, we present an exceptional example to challenge this postulate. MycG, a multifunctional biosynthetic P450 monooxygenase responsible for hydroxylation and epoxidation of 16-membered ring macrolide mycinamicins, is shown to catalyze the unnatural N-demethylation(s) of a range of mycinamicin substrates when partnered with the free Rhodococcus reductase domain RhFRED or the engineered Rhodococcus-spinach hybrid reductase RhFRED-Fdx. By contrast, MycG fused with the RhFRED or RhFRED-Fdx reductase domain mediates only physiological oxidations. This finding highlights the larger potential role of variant redox partner protein–protein interactions in modulating the catalytic activity of P450 enzymes.

The protein amide (1)H(N) chemical shift temperature coefficient reflects thermal expansion of the N-H...O=C hydrogen bond

The protein amide 1HN chemical shift temperature coefficient can be determined with high accuracy by recording spectra at different temperatures, but the physical mechanism responsible for this temperature dependence is not well understood. In this work, we find that this coefficient strongly correlates with the temperature coefficient of the through-hydrogen-bond coupling, 3hJNC′, based on NMR measurements of protein GB3. Parallel tempering molecular dynamics simulation suggests that the hydrogen bond distance variation at different temperatures/replicas is largely responsible for the 1HN chemical shift temperature dependence, from which an empirical equation is proposed to predict the hydrogen bond thermal expansion coefficient, revealing responses of individual hydrogen bonds to temperature changes. Different expansion patterns have been observed for various networks formed by β strands.

Aeration and mass transfer optimization in a rectangular airlift loop photobioreactor for the production of microalgae

Effects of superficial gas velocity and top clearance on gas holdup, liquid circulation velocity, mixing time, and mass transfer coefficient are investigated in a new airlift loop photobioreactor (PBR), and empirical models for its rational control and scale-up are proposed. In addition, the impact of top clearance on hydrodynamics, especially on the gas holdup in the internal airlift loop reactor, is clarified; a novel volume expansion technique is developed to determine the low gas holdup in the PBR. Moreover, a model strain of Chlorella vulgaris is cultivated in the PBR and the volumetric power is analyzed with a classic model, and then the aeration is optimized. It shows that the designed PBR, a cost-effective reactor, is promising for the mass cultivation of microalgae.

NMR detection of bifurcated hydrogen bonds in large proteins.

Hydrogen bonds play critical roles in protein structure, stability, and function. Conventionally, hydrogen bonds are mainly determined by X-ray crystallography and NOE-based NMR spectroscopy in indirect manners. In recent years, it was demonstrated that hydrogen bonds can be directly detected through NMR measurements of trans-hydrogen-bond scalar coupling constants. Here we report across hydrogen-bond protium/deuterium isotope effects in a 35 kDA protein observed with the isotopomer-selective TROSY NMR technique (Liu et al. J. Biomol. NMR 2006, 36, 205−214; Liu et al. J. Magn. Reson. 2007, 186, 319−326) and show that such isotope effects can be used to detect a most common type of bifurcated hydrogen bonds, in which a heavy atom, usually oxygen, is involved in two hydrogen bonds, including a pair of bifurcated hydrogen bonds involving a bound water molecule.

Chemical shift anisotropy of imino 15N nuclei in Watson-Crick base pairs from magic angle spinning liquid crystal NMR and nuclear spin relaxation

Knowledge of (15)N chemical shift anisotropy is prerequisite both for quantitative interpretation of nuclear spin relaxation rates in terms of local dynamics and for the use of residual chemical shift anisotropy (RCSA) as a constraint in structure determination. Accurate measurement of the very small RCSA from the difference in (15)N chemical shift under isotropic and weakly aligning liquid crystalline conditions is very sensitive to minute differences in sample conditions, such as pH or ionic strength. For this reason, chemical shifts were measured for the same solution, under static liquid crystalline alignment, and under magic angle spinning conditions where alignment relative to the magnetic field is removed. Measurements were made for 14 well-resolved G-N(1) and 6 U-N(3) (15)N nuclei in a sample of tRNA(Val). Fitting these RCSA data together with (15)N-(1)H dipole-CSA cross-correlated relaxation measurements to the recently refined structural model of tRNA(Val) yields the magnitude, asymmetry, and orientation of the (15)N CSA tensors.

Engineering a More Thermostable Blue Light Photo Receptor Bacillus subtilis YtvA LOV Domain by a Computer Aided Rational Design Method

The ability to design thermostable proteins offers enormous potential for the development of novel protein bioreagents. In this work, a combined computational and experimental method was developed to increase the T m of the flavin mononucleotide based fluorescent protein Bacillus Subtilis YtvA LOV domain by 31 Celsius, thus extending its applicability in thermophilic systems. Briefly, the method includes five steps, the single mutant computer screening to identify thermostable mutant candidates, the experimental evaluation to confirm the positive selections, the computational redesign around the thermostable mutation regions, the experimental reevaluation and finally the multiple mutations combination. The adopted method is simple and effective, can be applied to other important proteins where other methods have difficulties, and therefore provides a new tool to improve protein thermostability.