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PubMed1997

Effect of a single ethanol exposure on HIV replication in human lymphocytes.

BACKGROUND: Alcoholism is known to cause perturbations in cellular and humoral immunity, and some data suggest that acute alcohol ingestion enhances HIV replication in the lymphocytes of drinkers. METHODS: To study the acute effects of alcohol ingestion on HIV replication, oral ethanol (1 g/kg) was administered to 12 healthy volunteers in a controlled clinical setting. In vitro replication of HIV in the subjects' cultured lymphocytes and changes in lymphocyte phenotypes were evaluated. RESULTS: Statistically significant increases in peripheral lymphocytes and natural killer cell numbers were identified after the initial ethanol trial. HIV replication also increased in the isolated lymphocytes of some subjects after ethanol ingestion, but most subjects in the second trial showed essentially no changes in any of these parameters. CONCLUSIONS: Our results are consistent with either a subtle, study-induced stress-related enhancement in HIV replication or significant individual variation in response to ethanol. The results do not provide evidence for a general increase in HIV replication in the lymphocytes of subjects following a single in vivo ethanol dose of 1 g/kg.

Adult↗
PubMed1977

Fluorescent probes in model membranes I: anthroyl fatty acid derivatives in monolayers and liposomes of dipalmitoylphosphatidylcholine.

A study of three fluorescent anthroyl probes has been carried out using pure and mixed monomolecular films with dipalmitoylphosphatidylcholine. In addition, fluorescence depolarization and differential scanning calorimetry data were obtained from dipalmitoylphosphatidylcholine vesicles with incorporated anthroyl probes. The three probes used were 2-(9-anthroyl)palmitic acid. 12-(9-anthroyl)stearic acid, and 16-(9-anthroyl)palmitic acid. The latter probe was synthesized for these studies. In monolayers the probes shifted the onset of the liquid-condensed/liquid-expanded monolayer phase transition with the extent of the shift decreasing in the order: 2-(9-anthroyl)palmitic acid greater than 12-(9-anthroyl)stearic acid greater than 16-(9-anthroyl)stearic acid. A corresponding decrease in the gel-liquid crystalline bilayer transition temperature (Tc) showed the same order of perturbation in both the fluorescence depolarization and differential scanning calorimetry data. Locating the anthroyl entity in the center of the bilayer would appear to provide a minimum perturbation.

Anthracenes↗
PubMed1994

Perturbation of a prehension movement in Parkinson's disease.

Movement kinematics of the transport and manipulation components of a double-step prehension task were studied in eight Parkinson patients and eight control subjects. The aims were to (a) assess the effects of a spatial perturbation upon the movement for the two groups and (b) add data to the controversy about the damage/preservation of predictive behaviour in Parkinson patients. The results showed: (a) Both groups are able to preprogram a movement. (b) In both groups, the perturbation results in an anticipation of all kinematic parameters, both of the transport and manipulation components. (c) Parkinson patients, when adopting a predictive behavior, show a delay between the beginning of the two components, and thus activate them in sequence rather than simultaneously. This delay is significantly reduced by the perturbation, indicating that Parkinson patients, when using a responsive behavior, can recouple the two motor components.

Aged↗
PubMed1992

A network model of respiratory rhythmogenesis.

A mathematical model of the three-phase respiratory network proposed by Richter et al. (News Physiol. Sci. 1: 109-112, 1986) is developed and its properties are examined. The model reproduces the experimentally determined trajectories of membrane potential for the five physiologically distinct types of neurons included. Stepwise parameter changes can produce a respiratory rhythm with only two separate electrophysiological phases, result in apnea, or produce more complex patterns of firing. The phase-resetting behavior of the model was obtained with perturbing stimuli and is comparable to experimentally determined phase-resetting data. There is reasonable agreement between model predictions and experimental results. In the model, the properties of the phase singularity make termination of the respiratory rhythm by an appropriately timed perturbation virtually impossible, which is in agreement with experimental observations. The rhythm can be stopped by alterations that simulate the effect of input from the superior laryngeal nerve; the rhythm is locked in the postinspiratory phase. We conclude that our results are consistent with the concept of a network oscillator as the source of the respiratory rhythm.

Animals↗
PubMed2001

Neuromuscular Response Characteristics in Men and Women After Knee Perturbation in a Single-Leg, Weight-Bearing Stance.

OBJECTIVE: We examined whether muscle response times and activation patterns in the lower extremity differed between men and women in response to a rotational knee perturbation while standing in a single-leg, weight-bearing stance. DESIGN AND SETTING: We used a lower extremity perturbation device to produce a sudden, forward, and either internal or external rotation moment of the trunk and femur relative to the weight-bearing tibia. Subjects completed 10 trials of both internal and external rotation perturbation; the first 5 acceptable trials were averaged and used for data analysis. Two separate, repeated-measures analyses of variance were used to compare myoelectric response times and activation patterns between men and women for both internal and external rotation perturbation. SUBJECTS: Thirty-two female (19 lacrosse, 13 soccer) and 32 male (lacrosse) healthy intercollegiate athletes participated in the study. MEASUREMENTS: We used surface electromyography to record long latency reflex times of the medial and lateral quadriceps, hamstring, and gastrocnemius muscles. RESULTS: Women responded faster than men, primarily due to a shorter latency in quadriceps activation. However, men and women exhibited no difference in muscle-recruitment order. CONCLUSIONS: Although men and women demonstrated similar muscle-recruitment patterns to an imposed lower extremity perturbation, women tended to activate their quadriceps earlier than men. Whether this earlier quadriceps activation diminishes the ability of the hamstrings to adequately stabilize the knee joint or subjects the anterior cruciate ligament to greater risk of injury is still unknown and requires further study. Furthermore, although surface electromyography and measurement of myoelectric response times are useful in evaluating the timing, activation order, and coactivity patterns of the knee musculature, future studies should evaluate sex differences across the complete response continuum, including measures of intrinsic muscle stiffness, electromechanical delay, and time to force production.

Journal Article↗
PubMed2005

Parameterization of a nonlinear genotype to phenotype map using molecular networks.

Mathematical models of networks of molecular interactions controlling the expression of traits could theoretically be used as genotype to phenotype (GP) maps. Such maps are nonlinear functions of the environment and the genotype. It is possible to use nonlinear least square minimization methods to fit a model to a set of phenotypic data but the convergence of these methods is not automatic and may lead to a multiplicity of solutions. Both factors raise a number of questions with respect to using molecular networks as nonlinear maps. A method to fit a molecular network representing a bistable switch to various types of phenotypic data is introduced. This method relies on the identification of the model stable steady states and the estimation of the proportion of cells in each of them. By using environmental perturbations, it is possible to collect time-series of phenotypic data resulting in a smooth objective function leading to a good estimate of the parameters used to generate the simulated phenotypes.

Chromosome Mapping↗
PubMedDate not supplied

Covalent carcinogenic lesions in DNA: NMR studies of O6-methylguanosine containing oligonucleotide duplexes.

We report on proton and phosphorus high resolution NMR investigations of the self-complementary dodecanucleotide d(C1-G2-N3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplexes (henceforth called O6 meG.N 12-mers), N = C, T, A and G, which contain N3.O6meG10 interactions in the interior of the helix. These sequences containing a single modified O6meG per strand were prepared by phosphoamidite synthesis and provide an excellent model for probing the structural basis for covalent carcinogenic lesions in DNA. Distance dependent nuclear Overhauser effect (NOE) measurements and line widths of imino protons demonstrate that the N3 and O6meG.10 bases stack into the duplex and are flanked by stable Watson-Crick base pairs at low temperature for all four O6meG.N 12-mer duplexes. The imino proton of T3 in the O6meG.T 12-mer and G3 in the O6meG.N 12-mer helix, which are associated with the modification site, resonate at unusually high field (8.5 to 9.0 ppm) compared to imino protons in Watson-Crick base pairs (12.5 to 14.5 ppm). The nonexchangeable base and sugar protons have been assigned from two dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) measurements on the O6meG.N 12-mer helices. The directionality of the distance dependent NOEs establish all O6meG.N duplexes to be right-handed helices in solution. The glycosidic torsion angles are in the anti range at the N3.O6meG10 modification site except for O6meG10 in the O6meG.G 12-mer duplex which adopts a syn configuration. This results in altered NOEs between the G3 (anti).O6meG10 (syn) pair and flanking G2.C11 and G4.C9 base pairs in the O6meG.G 12-mer duplex. We observe pattern reversal for cross peaks in the COSY spectrum linking the sugar H1' protons with the H2',2" protons at the G2 and O6meG10 residues in the O6meG.N 12-mer duplexes with the effect least pronounced for the O6meG.T 12-mer helix. The proton chemical shift and NOE data have been analyzed to identify regions of conformational perturbations associated with N3.O6meG10 modification sites in the O6meG.N 12-mer duplexes. The proton decoupled phosphorus spectrum of O6meG.T 12-mer duplex exhibits an unperturbed phosphodiester backbone in contrast to the phosphorus spectra of the O6meG.C 12-mer, O6meG.G 12-mer and O6meG.A 12-mer duplexes which exhibit phosphorus resonances dispersed over 2 ppm characteristic of altered phosphodiester backbones at the modification site. Tentative proposals are put forward for N3.O6meG10 pairing models based on the available NMR data and serve as a guide for the design of future experiments.

Base Sequence↗
PubMed1997

The stability of insulin in crystalline and amorphous solids: observation of greater stability for the amorphous form.

PURPOSE: Generalizations based upon behavior of small molecules have established that a crystalline solid is generally much more stable toward chemical degradation than is the amorphous solid. This study examines the validity of this generalization for proteins using biosynthetic human insulin as the model protein. METHODS: Amorphous insulin was prepared by freeze drying the supernate from a suspension of zinc insulin crystals adjusted to pH 7.1. Storage stability at 25 degrees C and 40 degrees C were compared for the freeze dried material, the dried suspended crystals, and the starting batch of crystals. Samples were equilibrated at selected relative humidities between zero and 75% to obtain samples at various water contents. Assays for dimer formation were performed by size exclusion HPLC and assays for deamidated product were carried out by reverse phase HPLC. Degradation was found to be linear in square root of time, and the slopes from % degradation vs. square root of time were used to define the rate constants for degradation. Differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR) were used to characterize the state of the protein in the solids. RESULTS: As expected based upon previous results, the primary degradation pathways involve deamidation at the AsnA21 site and co-valent dimer formation, presumably involving the A-21 site. Contrary to expectations, amorphous insulin is far more stable than crystalline insulin under all conditions investigated. While increasing water content increases the rate of degradation of crystalline insulin, rate constants for degradation in the amorphous solid are essentially independent of water content up to the maximum water content studied (approximately 15%). CONCLUSIONS: Based upon the FTIR and DSC data, both crystalline and amorphous insulin retain some higher order structure when dried, but the secondary structure is significantly perturbed from that characteristic of the native solution state. However, neither DSC nor FTIR data provide a clear interpretation of the difference in stability between the amorphous and crystalline solids. The mechanism responsible for the superior stability of amorphous insulin remains obscure.

Calorimetry, Differential Scanning↗
PubMed2005

A new type of gradiometer for the receiving circuit of magnetic induction tomography (MIT).

Magnetic induction tomography (MIT) is a low-resolution imaging modality which aims at the three-dimensional (3D) reconstruction of the electrical conductivity in objects from alternating magnetic fields. In MIT systems the magnetic field perturbations to be detected are very small when compared to the excitation field (ppm range). The voltage which is induced by the excitation field in the receiver coils must be suppressed for providing sufficient dynamic range. In the past, two very efficient strategies were proposed: adjusted planar gradiometers (PGRAD) and the orientation of a receiver coil with respect to the excitation coil such that the net magnetic flow is zero (zero flow coil, ZFC). In contrast to the PGRAD no voltage is induced in the ZFC by the main field. This is advantageous because two comparatively high voltages in the two gradiometer coils can never be subtracted perfectly, thus leaving a residual voltage which is prone to drift. However, a disadvantage of the ZFC is the higher susceptibility to interferences from far RF sources. In contrast, in the gradiometer such interferences are cancelled to a high degree. We developed a new type of gradiometer (zero flow gradiometer, ZFGRAD) which combines the advantages of ZFC and PGRAD. All three systems were compared with respect to sensitivity and perturbation to signal ratio (PSR) defined as the ratio of the signal change due to a magnetic perturbation field at the carrier frequency and the signal change due to shifting a metallic sphere between two test points. The spatial sensitivity of the three systems was found to be very similar. The PSR of the ZFGRAD was more than 12 times lower than that of the ZFC. Finally, the feasibility of image reconstruction with two arrays of eight excitation coils and eight ZFGRAD, respectively, was shown with a single-step Gauss-Newton reconstructor and simulated measurement data generated for a cylindrical tank with a spherical perturbation. The resulting images show a clear, bright feature at the correct position of the sphere and are comparable to those with PGRAD arrays.

Animals↗
PubMedDate not supplied

Dynamical stability in the acquisition and performance of rhythmic ball manipulation: theoretical insights with a clinical slant.

Three experiments illustrate how a task-based approach and dynamical modeling of a perceptual-motor task can provide a useful framework for understanding functional and dysfunctional behavior. In the chosen task, subjects held a racket and bounced a ball rhythmically in the air with invariant ball amplitude. As such, the task could be cast into a mechanical model that encompassed the movements of the actor (racket) and the manipulandum (ball). In this form, the movement task is a dynamical system that displays dynamical stability, i.e., performance where perturbations die out by themselves. The hypothesis is that skilled actors seek to perform with this "passive" stability as it alleviates the control demands because perturbations do not require explicit corrections. In the experimental data, this strategy could be characterized by a single parameter, the acceleration of the racket at impact, which provided quantitative predictions. Experiment 1 established that subjects with normal sensorimotor functions indeed performed with racket acceleration values that were predicted to provide passive stability. Experiment 2 showed that subjects improved their skill over a course of 40 practice trials, as evidenced in decreased variability and accompanied by a change in racket acceleration toward values that provided optimal stability. In experiment 3, perturbations were applied and the subjects' adaptability was tested. When perturbations were large enough, subjects altered their racket timing to resume contact that provided stability. The results are discussed with their relevance to clinical contexts: How can such a task-based approach provide insights into the control of functional and dysfunctional movements? Can such behavioral results serve as a diagnostic tool? How can this approach to sensorimotor behavior stimulate physicians and therapists to progress therapeutic measures?

Biomechanical Phenomena↗
PubMed2005

Characterizing the amplitude dynamics of the human core-temperature circadian rhythm using a stochastic-dynamic model.

Two measures, amplitude and phase, have been used to describe the characteristics of the endogenous human circadian pacemaker, a biological clock located in the hypothalamus. Although many studies of change in circadian phase with respect to different stimuli have been conducted, the physiologic implications of the amplitude changes (dynamics) of the pacemaker are unknown. It is known that phase changes of the human circadian pacemaker have a significant impact on sleep timing and content, hormone secretion, subjective alertness and neurobehavioral performance. However, the changes in circadian amplitude with respect to different stimuli are less well documented. Although amplitude dynamics of the human circadian pacemaker are observed in physiological rhythms such as plasma cortisol, plasma melatonin and core temperature data, currently methods are not available to accurately characterize the amplitude dynamics from these rhythms. Of the three rhythms core temperature is the only reliable variable that can be monitored continuously in real time with a high sampling rate. To characterize the amplitude dynamics of the circadian pacemaker we propose a stochastic-dynamic model of core temperature data that contains both stochastic and dynamic characteristics. In this model the circadian component that has a dynamic characteristic is represented as a perturbation solution of the van der Pol equation and the thermoregulatory response in the data that has a stochastic characteristic is represented as a first-order autoregressive process. The model parameters are estimated using data with a maximum likelihood procedure and the goodness-of-fit measures along with the associated standard error of the estimated parameters provided inference about the amplitude dynamics of the pacemaker. Using this model we analysed core temperature data from an experiment designed to exhibit amplitude dynamics. We found that the circadian pacemaker recovers slowly to an equilibrium level following amplitude suppression. In humans this reaction to perturbation from equilibrium value has potential physiological implications.

Body Temperature Regulation↗
PubMed1980

Device for measuring the precision of eye-hand coordination while tracking changing size.

Psychophysical evidence supports the idea that the human visual pathway computes an object's rate of change of angular size rather independently of the object's trajectory and rather independently of other visual parameters, including contrast and intensity. This independence could provide a basis for accurately judging the component of an object's velocity along a line through the eye in the working visual environment where many visual parameters vary simultaneously. We describe a procedure for quantifying a subject's ability to track changing size, and illustrate the procedure with preliminary experimental data. The subject's R.M.S. tracking errors are displayed in three frequency bands. Our device also measures the perturbing effect of sideways motion upon the subject's ability to track changing size. Such data may go some way to predict a subject's performance in tasks of eye-limb coordination, especially where visual information is largely restricted to the changing-size channel.

Aerospace Medicine↗
PubMed1999

Modulating electron density in the bound product, 4-hydroxybenzoyl-CoA, by mutations in 4-chlorobenzoyl-CoA dehalogenase near the 4-hydroxy group.

The enzyme 4-chlorobenzoyl-CoA dehalogenase hydrolyzes 4-chlorobenzoyl-CoA (4-CBA-CoA) to 4-hydroxybenzoyl-CoA (4-HBA-CoA). Biochemical and crystallographic studies have identified a critical role for the dehalogenase residue Asp 145 in close proximity to the ligand's 4-hydroxy group in the structure of the product-enzyme complex. In the present study the effects of site selective mutations at Asp 145 on the product complex are explored by Raman spectroscopy. The spectral signatures of the WT-product complex, the large red shift in lambdamax, and the complete reorganization of the benzoyl ring modes in Raman data are absent for the D145E complex. The major spectral perturbations in the WT complex are brought about by strong electron "pull" at the benzoyl carbonyl and electron "push" by the side chain of Asp 145 near the 4-OH group. Acting in concert, these factors polarize the benzoyl's pi-electrons. Since the Raman data show that very strong electron pull occurs at the benzoyl's carbonyl in the D145E complex, it is apparent that the needed electron push near the benzoyl's 4-OH group is missing. Thus, very precise positioning of Asp 145's side chain near the benzoyl's 4-position is needed to bring about the dramatic electron reorganization seen in the WT complex, and this criterion cannot be met by the glutamate side chain with its additional CH2 group. For two other Asp145 mutants D145A and D145S that lack catalytic activity, Raman difference spectroscopic data for product complexes demonstrate the presence of a population of ionized product (i.e., 4-O-) in the active sites. The presence of the ionized phenolate form explains the observation that these complexes have highly red-shifted absorbance maxima with lambdamaxs near 400 nm. For the WT complex only the 4-OH form is seen, ionization being energetically expensive with the presence of the proximal negative charge on the Asp 145 side chain. Semiquantitative estimates of the pKa for the bound product in D145S and D145A indicate that this ionization lies in the pH 6.5-7.0 range. This is approximately 2 pH units below the pKa for the free product. The Raman spectrum of 4-dimethylaminobenzoyl-CoA undergoes major changes upon binding to dehalogenase. The bound form has two features near 1562 and 1529 cm-1 and therefore closely resembles the spectrum of product bound to wild-type enzyme, which underlines the quinonoid nature in these complexes. The use of a newly developed Raman system allowed us to obtain normal (nonresonance) Raman data for the dehalogenase complexes in the 100-300 microM range and heralds an important advance in the application of Raman spectroscopy to dilute solutions of macromolecules.

Acyl Coenzyme A↗
PubMed1995

A recurring dominant negative mutation causes autosomal dominant growth hormone deficiency--a clinical research center study.

Familial isolated GH deficiency type II (IGHD-II) is an autosomal dominant disorder that has been previously shown in some patients to be caused by heterogeneous GH gene defects that affect GH messenger RNA (mRNA) splicing. We report here our finding of multiple G-->A transitions of the first base of the donor splice site of IVS 3 (+ 1G-->A) in IGHD II subjects from three nonrelated kindreds from Sweden, North America, and South Africa. This + 1G-->A substitution creates an NlaIII site that was used to demonstrate that all affected individuals in all three families were heterozygous for the mutation. To determine the effect of this mutation on GH mRNA processing, HeLa cells were transfected with expression plasmids containing normal or mutant + 1G-->A alleles, and complementary DNAs from the resulting GH mRNAs were sequenced. The mutation was found to destroy the GH IVS 3 donor splice site, causing skipping of exon 3 and loss of the codons for amino acids 32-71 of the mature GH peptide from the mutant GH mRNA. Our finding of exon 3 skipping in transcripts of the + 1G-->A mutant allele is identical to our previous report of a different sixth base transition (+6T-->C) mutation of the IVS 3 donor splice site that also causes IGHD II. Microsatellite analysis of an affected subjects' DNA from each of the three nonrelated kindreds indicates that the + 1G-->A mutation arose independently in each family. Finding that neither grandparent has the mutation in the first family suggests that it arose de novo in that family. Our data indicate that 1) + 1G-->A IVS 3 mutations perturb GH mRNA splicing and cause IGHD II; and 2) these mutations can present as de novo GHD cases.

Adult↗
PubMed1986

Frequency and amplitude perturbation analysis of electroglottograph during sustained phonation.

Electroglottography (EGG) was used to monitor vocal fold vibration patterns in normal subjects and patients with various laryngeal disorders. In order to evaluate the regularity of vocal fold vibration, frequency and amplitude perturbation of EGG waves during sustained phonation were measured with a laboratory computer. The data were compared to the degree of hoarseness evaluated by auditory perception and by sound spectrographic analysis. Frequency and amplitude perturbation measures showed some overlap between normal and pathological groups. However, there was a close relation between perturbation analysis of EGG waves and degree of hoarseness (Spearman's rank correlation coefficient rs = 0.73, p less than 0.0005). Amplitude perturbation was found to be a more sensitive measure of the irregularity of vocal fold vibration than frequency perturbation.

Adolescent↗
PubMed2002

Perturbation of microtubule polymerization by quercetin through tubulin binding: a novel mechanism of its antiproliferative activity.

The dietary flavonoid quercetin has a broad range of biological activities, including potent antitumor activity against several types of tumors. Recently, it has been shown that quercetin inhibits cancer cells proliferation by depleting cellular microtubules and perturbing cellular microtubule functions. However, the direct interactions of quercetin with tubulin and microtubules have not been examined so far. Here, we found that quercetin inhibited polymerization of microtubules and depolymerized microtubules made from purified tubulin in vitro. The binding of quercetin with tubulin was studied using quercetin fluorescence and intrinsic tryptophan fluorescence of tubulin. Quercetin bound to tubulin at a single site with a dissociation constant of 5-7 microM, and it specifically inhibited colchicine binding to tubulin but did not bind at the vinblastine site. In addition, quercetin perturbed the secondary structure of tubulin, and the binding of quercetin stimulated the intrinsic GTPase activity of soluble tubulin. Further, quercetin stabilized tubulin against decay and protected two cysteine residues of tubulin toward chemical modification by 5,5'-dithiobis-2-nitrobenzoic acid. Our data demonstrated that the binding of quercetin to tubulin induces conformational changes in tubulin and a mechanism through which quercetin could perturb microtubule polymerization dynamics has been proposed. The data suggest that quercetin inhibits cancer cells proliferation at least in part by perturbing microtubule functions through tubulin binding.

Anilino Naphthalenesulfonates↗
PubMed2006

Study of four genes belonging to the folate pathway: transcobalamin 2 is involved in the onset of non-syndromic cleft lip with or without cleft palate.

Cleft lip with or without cleft palate (CL/P) is the most common inborn craniofacial anomaly. Affected individuals require extensive medical and psychosocial support. Although CL/P has a complex and poorly understood etiology, increasing evidence of folate pathway involvement has been collected. So far, only the MTHFR gene has been extensively investigated as a risk factor for CL/P, while little has been done to test genetic variations in the folate biosynthetic pathways that may influence the infant's susceptibility to these birth defects. To date, this paper presents the first attempt to verify the involvement of four genes belonging to the folate pathway in nonsyndromic cleft onset. We used a case-parent triad design to test for linkage disequilibrium in the case of seven SNPs mapping on four different genes: transcobalamin 1 and 2 (TCN1 and TCN2), methionine synthase (MTR), and MTR reductase (MTRR). Our finding suggests that TCN2 is involved in causing CL/P. Indeed, significant overtransmission of the C allele was observed at the polymorphism c.776C>G (p.Pro259Arg) to the affected offspring (P=0.01). Results obtained with additional TCN2 polymorphisms suggest that c.776C>G may be functionally related to CL/P. However, because conflicting data exist with regard to the effect of the polymorphism in transcobalamin 2 function or in perturbing plasma levels of key molecules in the folate pathway, further investigation is warranted to confirm our data.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed2004

Global analysis of three-state protein unfolding data.

A new method for analyzing three-state protein unfolding equilibria is described that overcomes the difficulties created by direct effects of denaturants on circular dichroism (CD) and fluorescence spectra of the intermediate state. The procedure begins with a singular value analysis of the data matrix to determine the number of contributing species and perturbations. This result is used to choose a fitting model and remove all spectra from the fitting equation. Because the fitting model is a product of a matrix function which is nonlinear in the thermodynamic parameters and a matrix that is linear in the parameters that specify component spectra, the problem is solved with a variable projection algorithm. Advantages of this procedure are perturbation spectra do not have to be estimated before fitting, arbitrary assumptions about magnitudes of parameters that describe the intermediate state are not required, and multiple experiments involving different spectroscopic techniques can be simultaneously analyzed. Two tests of this method were performed: First, simulated three-state data were analyzed, and the original and recovered thermodynamic parameters agreed within one standard error, whereas recovered and original component spectra agreed within 0.5%. Second, guanidine-induced unfolding titrations of the human retinoid-X-receptor ligand-binding domain were analyzed according to a three-state model. The standard unfolding free energy changes in the absence of guanidine and the guanidine concentrations at zero free-energy change for both transitions were determined from a joint analysis of fluorescence and CD spectra. Realistic spectra of the three protein states were also obtained.

Animals↗