The structure and activity of fungal lipases in bile-salt solutions

The changes in the structure and catalytic properties of fungal lipases (Candida rugosa, Rhizomucor miehei, Mucor javanicus) were investigated in micellar solutions of bile salts that differ in their hydrophilic–lypophilic balance and reaction medium properties. The methods of circular dichroism and tryptophan fluorescence were applied to estimate the changes in peptide structure within complexes with bile-salt micelles. Bile salts do not exert a significant influence on the structure of the enzymes under study: in the Rh. miehei and M. javanicus lipases the α-helix content was slightly decreased; an influence of the bile salts on the C. rugosa structure was not revealed. Despite negligible structural modifications in the enzymes, a considerable change in their catalytic properties, namely an abrupt decrease in catalytic effectiveness was observed in bile-salt solutions. Substrate–bile salt micelle complex formation was demonstrated by the NMR self-diffusion method. A model of the regulation of fungal lipase activity was proposed.     

Comparison of (13)C(alpha)H and (15)NH backbone dynamics in protein GB1

This study presents a site-resolved experimental view of backbone C(alpha)H and NH internal motions in the 56-residue immunoglobulin-binding domain of streptococcal protein G, GB1. Using (13)C(alpha)H and (15)NH NMR relaxation data [T(1), T(2), and NOE] acquired at three resonance frequencies ((1)H frequencies of 500, 600, and 800 MHz), spectral density functions were calculated as F(omega) = 2omegaJ(omega) to provide a model-independent way to visualize and analyze internal motional correlation time distributions for backbone groups in GB1. Line broadening in F(omega) curves indicates the presence of nanosecond time scale internal motions (0.8 to 5 nsec) for all C(alpha)H and NH groups. Deconvolution of F(omega) curves effectively separates overall tumbling and internal motional correlation time distributions to yield more accurate order parameters than determined by using standard model free approaches. Compared to NH groups, C(alpha)H internal motions are more broadly distributed on the nanosecond time scale, and larger C(alpha)H order parameters are related to correlated bond rotations for C(alpha)H fluctuations. Motional parameters for NH groups are more structurally correlated, with NH order parameters, for example, being larger for residues in more structured regions of beta-sheet and helix and generally smaller for residues in the loop and turns. This is most likely related to the observation that NH order parameters are correlated to hydrogen bonding. This study contributes to the general understanding of protein dynamics and exemplifies an alternative and easier way to analyze NMR relaxation data.     

Heat capacities and a snapshot of the energy landscape in protein GB1 from the pre-denaturation temperature dependence of backbone NH nanosecond fluctuations

Protein stability is usually characterized calorimetrically by a melting temperature and related thermodynamic parameters. Despite its importance, the microscopic origin of the melting transition and the relationship between thermodynamic stability and dynamics remains a mystery. Here, NMR relaxation parameters were acquired for backbone 15NH groups of the 56 residue immunoglobulin-binding domain of streptococcal protein G over a pre-denaturation temperature range of 5-50 degrees C. Relaxation data were analyzed using three methods: the standard three-Lorentzian model free approach; the F(omega)=2omegaJ(omega) spectral density approach that yields motional correlation time distributions, and a new approach that determines frequency-dependent order parameters. Regardless of the method of analysis, the temperature dependence of internal motional correlation times and order parameters is essentially the same. Nanosecond time-scale internal motions are found for all NHs in the protein, and their temperature dependence yields activation energies ranging up to about 33kJ/mol residue. NH motional barrier heights are structurally correlated, with the largest energy barriers being found for residues in the most "rigid" segments of the fold: beta-strands 1 and 4 and the alpha-helix. Trends in this landscape also parallel the free energy of folding-unfolding derived from hydrogen-deuterium (H-D) exchange measurements, indicating that the energetics for internal motions occurring on the nanosecond time-scale mirror those occurring on the much slower time-scale of H-D exchange. Residual heat capacities, derived from the temperature dependence of order parameters, range from near zero to near 100J/mol K residue and correlate with this energy landscape. These results provide a unique picture of this protein's energy landscape and a relationship between thermodynamic stability and dynamics that suggests thermosensitive regions in the fold that could initiate the melting process.     

Internal motional amplitudes and correlated bond rotations in an alpha-helical peptide derived from 13C and 15N NMR relaxation

Peptide GFSKAELAKARAAKRGGY folds in an alpha-helical conformation that is stabilized by formation of a hydrophobic staple motif and an N-terminal capping box (Munoz V. Blanco FJ, Serrano L, 1995, Struct Biol 2:380-385). To investigate backbone and side-chain internal motions within the helix and hydrophobic staple, residues F2, A5, L7, A8, and A10 were selectively 13C- and 15N-enriched and NMR relaxation experiments were performed in water and in water/trifluoroethanol (TFE) solution at four Larmor frequencies (62.5, 125, 150, and 200 MHz for 13C). Relaxation data were analyzed using the model free approach and an anisotropic diffusion model. In water, angular variances of motional vectors range from 10 to 20 degrees and backbone phi,psi bond rotations for helix residues A5, L7, A8, and A10 are correlated indicating the presence of Calpha-H, Calpha-Cbeta, and N-H rocking-type motions along the helix dipole axis. L7 side-chain CbetaH2 and CgammaH motions are also correlated and as motionally restricted as backbone CalphaH, suggesting considerable steric hindrance with neighboring groups. In TFE which stabilizes the fold, internal motional amplitudes are attenuated and rotational correlations are increased. For the side chain of hydrophobic staple residue F2, wobbling-in-a-cone type motions dominate in water, whereas in TFE, the Cbeta-Cgamma bond and phenyl ring fluctuate more simply about the Calpha-Cbeta bond. These data support the Daragan-Mayo model of correlated bond rotations (Daragan VA, Mayo KH, 1996, J Phys Chem 100:8378-8388) and contribute to a general understanding of internal motions in peptides and proteins.     

A Method for Chronic Registration of Brain Cortical Electrical Activity in Rats

Journal of Evolutionary Biochemistry and Physiology - Electrocorticogram registration and analysis (electrocorticography, ECoG) is widely used in small-animal biomedical research. To date, a...     

Assessment of Dysmyelination with RAFFn MRI: Application to Murine MPS I

Type I mucopolysaccharidosis (MPS I) is an autosomal recessive lysosomal storage disorder with neurological features. Humans and laboratory animals with MPS I exhibit various white matter abnormalities involving the corpus callosum and other regions. In this study, we first validated a novel MRI technique, entitled Relaxation Along a Fictitious Field in the rotating frame of rank n (RAFFn), as a measure of myelination and dysmyelination in mice. We then examined differences between MPS I mice and heterozygotes using RAFF5 and histology. RAFF5 (i.e., RAFFn with n = 5) relaxation time constants were highly correlated with histological myelin density (R2 = 0.68, P<0.001), and RAFF5 clearly distinguished between the hypomyelinated and dysmyelinated shiverer mouse and the wild-type mouse. Bloch-McConnell theoretical analysis revealed slower exchange correlation times and smaller exchange-induced relaxation rate constants for RAFF4 and RAFF5 compared to RAFF1-3, T_1ρ, and T_2ρ. These data suggest that RAFF5 may assess methylene protons in myelin lipids and proteins, though other mechanisms (e.g. detection of myelin-bound water) may also explain the sensitivity of RAFF5 to myelin. In MPS I mice, mean RAFF5 relaxation time constants were significantly larger for the striatum (P = 0.004) and internal capsule (P = 0.039), and marginally larger for the fornix (P = 0.15). Histological assessment revealed no differences between MPS I mice and heterozygotes in myelin density or corpus callosum thickness. Taken together, these findings support subtle dysmyelination in the brains of mice with MPS I. Dysmyelination may result from myelin lipid abnormalities caused by the absence of α-L-iduronidase. Our findings may help to explain locomotor and cognitive deficits seen in mice with MPS I.     

The Folding of the Specific DNA Recognition Subdomain of the Sleeping Beauty Transposase Is Temperature-Dependent and Is Required for Its Binding to the Transposon DNA

The reaction of DNA transposition begins when the transposase enzyme binds to the transposon DNA. Sleeping Beauty is a member of the mariner family of DNA transposons. Although it is an important tool in genetic applications and has been adapted for human gene therapy, its molecular mechanism remains obscure. Here, we show that only the folded conformation of the specific DNA recognition subdomain of the Sleeping Beauty transposase, the PAI subdomain, binds to the transposon DNA. Furthermore, we show that the PAI subdomain is well folded at low temperatures, but the presence of unfolded conformation gradually increases at temperatures above 15°C, suggesting that the choice of temperature may be important for the optimal transposase activity. Overall, the results provide a molecular-level insight into the DNA recognition by the Sleeping Beauty transposase.     

Use of Naïve Bayes Classifier to Assess the Effects of Antipsychotic Agents on Brain Electrical Activity Parameters in Rats

Journal of Evolutionary Biochemistry and Physiology - Research and development of novel methods to determine the effects of antipsychotic agents is an important challenge for experimental...     

Conformational exchange on the microsecond time scale in alpha-helix and beta-hairpin peptides measured by 13C NMR transverse relaxation

13C-NMR relaxation experiments (T(1), T(2), T(1)(rho), and NOE) were performed on selectively enriched residues in two peptides, one hydrophobic staple alpha-helix-forming peptide GFSKAELAKARAAKRGGY and one beta-hairpin-forming peptide RGITVNGKTYGR, in water and in water/trifluoroethanol (TFE). Exchange contributions, R(ex), to spin-spin relaxation rates for (13)C(alpha) and (13)C(beta) groups were derived and were ascribed to be mainly due to peptide folding-unfolding. To evaluate the exchange time, tau(ex), from R(ex), the chemical shift difference between folded and unfolded states, Deltadelta, and the populations of these states, p(i), were determined from the temperature dependence of (13)C chemical shifts. For both peptides, values for tau(ex) fell in the 1 micros to 10 micros range. Under conditions where the peptides are most folded (water/TFE, 5 degrees C), tau(ex) values for all residues in each respective peptide were essentially the same, supporting the presence of a global folding-unfolding exchange process. Rounded-up average tau(ex) values were 4 micros for the helix peptide and 9 micros for the hairpin peptide. This 2-3-fold difference in exchange times between helix and hairpin peptides is consistent with that observed for folding-unfolding of other small peptides.