Changes in pro-inflammatory markers and leucine concentrations in response to Nordic Walking training combined with vitamin D supplementation in elderly women.

Mechanisms underpinning age-related decreases in muscle strength and muscle mass relate to chronic inflammation. Physical activity induces an anti-inflammatory effect, but it is modulated by additional factors. We hypothesized that vitamin D, which has also anti-inflammatory activity will modify adaptation to exercise and reduce inflammation in elderly women. Twenty-seven women aged 67 ± 8 years were included and divided into groups with baseline vitamin D concentration more than 20 ng mL-1 (MVD) and less than 20 ng mL-1 (LVD). Both groups performed 1 h Nordic Walking (NW) training combined with vitamin D supplementation for 12 weeks. Serum concentrations of inflammation markers, branched amino acids, vitamin D, muscle strength and balance were assessed at the baseline and three days after intervention. The training caused the significant decrease in concentration of pro-inflammatory proteins HMGB1 (30 ± 156%; 90% CI) and IL-6 (-10 ± 66%; 90% CI) in MVD group. This effects in group MVD were moderate, indicating vitamin D as one of the modifiers of these exercise-induced changes. Rise of myokine irisin induced by exercise correlated inversely with HMGB1 and the correlation was more pronounced at the baseline as well as after training among MVD participants. Although the intervention caused the leucine level to rise, a comparison of the recorded response between groups and the adjusted effect indicated that the effect was 20% lower in the LVD group. Overall the applied training program was effective in reducing HMGB1 concentration. This drop was accompanied by the rise of myokine irisin and better uptake of leucine among women with higher baseline vitamin D.

The effect of the competitive season in professional basketball on inflammation and iron metabolism.

Following acute physical activity, blood hepcidin concentration appears to increase in response to exercise-induced inflammation, but the long-term impact of exercise on hepcidin remains unclear. Here we investigated changes in hepcidin and the inflammation marker interleukin-6 to evaluate professional basketball players' response to a season of training and games. The analysis also included vitamin D (25(OH)D3) assessment, owing to its anti-inflammatory effects. Blood samples were collected for 14 players and 10 control non-athletes prior to and after the 8-month competitive season. Athletes' performance was assessed with the NBA efficiency score. At the baseline hepcidin correlated with blood ferritin (r = 0.61; 90% CL ±0.31), but at the end of the season this correlation was absent. Compared with the control subjects, athletes experienced clear large increases in hepcidin (50%; 90% CI 15-96%) and interleukin-6 (77%; 90% CI 35-131%) and a clear small decrease in vitamin D (-12%; 90% CI -20 to -3%) at the season completion. Correlations between change scores of these variables were unclear (r = -0.21 to 0.24, 90% CL ±0.5), but their uncertainty generally excluded strong relationships. Athletes were hence concluded to have experienced acute inflammation at the beginning but chronic inflammation at the end of the competitive season. At the same time, the moderate correlation between changes in vitamin D and players' performance (r = 0.43) was suggestive of its beneficial influence. Maintaining the appropriative concentration of vitamin D is thus necessary for basketball players' performance and efficiency. The assessment of hepcidin has proven to be useful in diagnosing inflammation in response to chronic exercise.

Hypoxic Ventilatory Reactivity in Experimental Diabetes.

Diabetes, apart from generalized neuropathy and microangiopathy, involves tissue hypoxia, which may drive chronic proinflammatory state. However, studies on the ventilatory control in diabetes are sparse and conflicting. In this study we examined the function and morphology of diabetic carotid bodies (CBs). Diabetes was evoked in Wistar rats with streptozotocin (70 mg/kg, i.p.). The acute hypoxic ventilatory responses (HVR) to 12 and 8 % O(2) were investigated in conscious untreated rats after 2 and 4 weeks in a plethysmographic chamber. CBs were dissected and subjected to morphologic investigations: (1) electron transmission microscopy for ultrastructure and (2) laser scanning confocal microscopy to visualize the microvascular bed in sections labeled with the lectin Griffonia simplicifolia-I (GSI), an endothelial cell marker, and fluorescein isothiocyanate (FITC). All findings were referenced to the normal healthy rats. We found that diabetes distinctly dampened the HVR. At the ultrastructural level, the diabetic CB displayed proliferation of connective tissue and neovascularization deranging the interglomal structure, and lengthening the O(2) diffusion path from capillaries to chemoreceptor cells. The chemoreceptor cells remained largely unchanged. The endothelial cell labeling confirmed the intensive angiopathy and the induction of microvessel growth. We conclude that diabetes hampers the chemical regulation of ventilation due to remodeling of CB parenchyma, which may facilitate chronic hypoxia and inflammation in the organ.

Development and aging are oxygen-dependent and correlate with VEGF and NOS along life span.

During development and aging, vascular remodeling represents a critical adaptive response to modifications in oxygen supply to tissues. Hypoxia inducible factor (HIF) has a crucial role and is modulated by oxygen levels, with an age-dependent response in neonates, adult, and aged people. ROS are generated under hypoxic conditions and the accumulation of free radicals during life reduces the ability of tissues to their removal. In this immunohistochemical study we investigated the presence and localization of VEGF and iNOS in human carotid bodies (CB) sampled at autopsy from three children (mean age - 2 years), four adult young subjects (mean age - 44.3 years), and four old subjects (mean age - 67.3 years). VEGF immunoreactivity was significantly enhanced in CB tissues from the children (7.2 ± 1.2%) and aged subjects (4.7 ± 1.7%) compared with the young adults (1.4 ± 0.7%). On the other hand, iNOS immunoreactivity was enhanced in CB tissues from the children (0.4 ± 0.04%) and young adult subjects (0.3 ± 0.02%) compared with the old subjects (0.2 ± 0.02%). Prevention of oxygen desaturation, reducing all causes of hypoxemia from neonatal life to aging would decrease the incidence of diseases in the elderly population with lifespan extension.

Running a 100-km ultra-marathon induces an inflammatory response but does not raise the level of the plasma iron-regulatory protein hepcidin.

AIM: Exercise may induce an inflammatory response that may lead to changes in iron metabolism. The aim of this study was to examine the relationship between the inflammation induced by a 100 km run and the level of hepcidin, which is a hormone regulating iron metabolism. METHODS: Six males, age 44.5±13.5 years, running 100 km. SETTING: the CRP protein, IL-6 and leucocyte count were measured as an index of inflammation. RESULTS: A 100 km run caused a progressive increase in blood IL-6 concentration, which reached the highest values after 75 km. Furthermore, an increase in levels of CRP, a marker of inflammation, was observed after the 100 km run and continued to increase after a 14 h recovery period. Leucocyte number and markers of muscle damage were significantly elevated after the 100 km run. This was accompanied by a decrease in transferrin saturation and an increase in blood haemoglobin and ferritin. Despite all these changes, the 100 km race did not affect blood hepcidin concentration either during the run or after a 14 h recovery period. CONCLUSION: The study shows that a 100 km run induces an inflammatory response but does not trigger changes in the blood hepcidin level. Thus it can be concluded that changes in IL-6 are not sufficient to increase the blood hepcidin level in runners.

Hypoxic ventilatory response in limited iron in the rat.

The study seeks to determine the role of iron in the ventilatory response to acute hypoxia in anesthetized, spontaneously breathing Wistar rats, using an experimental paradigm of chronic iron chelation. Since the hypoxic ventilatory response is generated by carotid body chemoreceptors, another objective of the study was to assess the hitherto unknown effects of iron chelation on carotid body ultrastructure. Minute ventilation and its tidal and frequency components' responses to acute 9% FiO2 were measured with plethysmography before and after iron chelation with ciclopirox olamine (CPX, 20 mg/kg, i.p.) for 7 days. Transmission electron microscopy was employed to assess the ultrastructure of carotid body tissue. We found that CPX pretreatment significantly decreased both resting and peak hypoxic ventilation in a range of 20-25%. Iron chelation caused degenerative changes in carotid body parenchyma, particularly affecting the chemoreceptor cell ultrastructure, consisting of cytoplasmic vacuolization, formation of lysosomes and multivesicular bodies, and damage to mitochondria. We report herein that inhibition of ventilatory responsiveness in limited iron is explicable by iron's role in maintaining carotid body ultrastructural viability rather than by emulation of hypoxic HIF-1alpha-mediated transduction pathway in chemoreceptor cells suggested by previous in vitro studies.

Reduced pulmonary function is age-dependent in the rat lung in normoxia.

BACKGROUND: Oxygen transport is optimized at the cellular level, since oxygen serves as the terminal electron acceptor in mitochondrial oxidative phosphorylation and several enzymatic processes require molecular oxygen as substrate. During development and aging, redundant cells and exhausted cells are eliminated, respectively, whereas others can adapt to the stressful environment and survive. OBJECTIVE: The study investigated the molecular mechanisms activated in the lung during normal aging, through the expression of hypoxia inducible factor (HIF), vascular endothelial growth factor (VEGF), p53, p66⊃Shc, putative cysteine protease (CPP32) and kinaseB-α phosphorylation (pIkB-α). MATERIAL AND METHODS: Twelve male Wistar rats divided into two age-groups, each consisting of 6 animals, 3 and 24 months old, were used. The rats were anesthetized with Nembutal (40 mg/kg, ip) and the lungs were excised from each rat and processed for TUNEL and Western blotting analyses. RESULTS: The expressions of p53, p66⊃Shc and CPP32 were significantly increased in the old normoxic rat lung specimens, when compared with the young ones. In parallel, expressions of VEGF and pIkBα were increased in old rather than young rats. CONCLUSIONS: Aging leads to increased expressions of p53, p66⊃Shc and CPP32, suggesting that apoptosis is in progress. At the same time, the lung tries to counteract apoptosis through the production of VEGF and pIkB-α to adapt itself to a stressful situation. The aging lung creates a life-support system in order to counteract the apoptotic process.

Obstructive sleep apnea and type 2 diabetes.

Type 2 diabetes and obstructive sleep apnea (OSA) are diseases with high prevalence and major public health impact. There is evidence that regular snoring and OSA are independently associated with alterations in glucose metabolism. Thus, OSA might be a risk factor for the development of type 2 diabetes. Possible causes might be intermittent hypoxia and sleep fragmentation, which are typical features of OSA. OSA might also be a reason of ineffective treatment of type 2 diabetes. There is further evidence that the treatment of OSA by continuous positive airway pressure (CPAP) therapy might correct metabolic abnormalities in glucose metabolism. It is assumed that this depends on therapy compliance to CPAP. On the other hand, there are also hints in the literature that type 2 diabetes per se might induce sleep apnea, especially in patients with autonomic neuropathy. Pathophysiological considerations open up new insights into that problem. Based on the current scientific data, clinicians have to be aware of the relations between the two diseases, both from the sleep medical and the diabetological point of view. The paper summarizes the most important issues concerning the different associations of OSA and type 2 diabetes.

A single oral intake of arginine does not affect performance during repeated Wingate anaerobic test.

AIM: The ergogenic effect of arginine has been demonstrated in research focusing on its intake before exercise. However, in these studies, the effect of arginine in combination with other various metabolites were assessed. The aim of this study was to determine whether a single oral intake of arginine, without any other compounds, 60 minutes prior to exercise, modifies performance and exercise metabolism during a repeated Wingate anaerobic test. METHODS: Six healthy, active, but not highly trained volunteers participated in the study. Subjects performed three 30s all-out supramaximal Wingate Anaerobic Tests (WAnTs) with 4 minute-interval rest periods between WAnTs. RESULTS: Arginine ingestion before exercise did not influence physical performance. Triple WAnTs resulted in a marked increase in white blood cell (WBC) count, lactate and ammonia concentrations, however there were no differences between arginine and the placebo trials. CONCLUSION: Our data indicated that 2 g of arginine ingested in a single dose, neither induced nitrite/nitrate (NOx) concentrations changes, nor improved physical performance.

Physiological carotid body denervation during aging.

Aging is characterized by a lower homeostatic capacity and the carotid body (CB) plays an important role during aging. Here, we sought to elucidate whether the aging effects on the oxygen-sensitive mechanisms in CB cells occur through a reduction of the contact surfaces in the synaptic junctions. The hypothesis was that the CB would undergo a "physiological denervation" in old age. Two groups of male Wistar rats, young (2-3 months old) and senescent (22 months old) were used. CBs were rapidly dissected and the specimens were subjected to a routine transmission electron microscopic procedure. Expressions of HIF-1 proportional, variant, VEGF and NOS-1 were evaluated by immunohistochemical analysis. Our results show that in the old CB, HIF-1 proportional, variant, VEGF and NOS-1 expressions decrease. The cell volume, the number of mitochondria and that of dense-cored vesicles were reduced, and the nucleus shrank. There also was an accumulation of lipofuscin and a proliferation of extracellular matrix. Most importantly, there were fewer synaptic connections between chemoreceptor cells. The total number of synapses observed in all electronograms decreased from 125 in the young to 28 in the old CB. These results suggest the aging CB undergoes a "physiological denervation" leading to a reduction in homeostatic capacity. The age-related reduction of synaptic junctions may be a self-protective mechanism through which cells buffer themselves against reactive oxygen species accumulation during aging.

NADH-generating substrates reduce peroxyl radical toxicity in RL-34 cells.

There is general agreement that oxidative stress may induce apoptotic and necrotic cell death. Recently it has been shown that NADH can be considered an important antioxidant as it reacts with peroxyl and alkoxyl radicals under in vitro conditions. Therefore, in the present study we hypothesized that an increase in intracellular NADH using specific substrates will protect RL-34 cells against cytotoxicity of 2'-azobis (2-amidinopropane) dihydrochloride (AAPH), which is a peroxyl radical generating compound. Cells treated for 24 hours with 6.0 mM AAPH were severely damaged: mitochondria were vacuolated, and the level of free radicals significantly increased. Both apoptotic and necrotic cells were detected (11.1% and 11.4%, respectively) even after 5 hours of treatment. Pretreatment of the cells with substrates which increase the intracellular level of NADH, such as lactate, beta-hydroxybutyrate, and ethanol, distinctly inhibited AAPH-induced reactive oxygen species (ROS) formation and cell death. On the other hand, acetoacetate (AcA), which decrease the intracellular level of NADH, had opposite effects. Interestingly, NADH-generating substrates augment, while AcA reduced superoxide radical formation induced by AAPH. These results may suggest that although NADH generating substrates may exert some deleterious effects within a cell by inducing reductive stress, they diminish alkoxyl or peroxyl radical cytotoxicity. The protection is associated with a decrease in ROS formation measured by dichlorofluorescein, but with an increase in superoxide radical formation.

Diffusional Encounter Rate Constants for Xanthone and 2-Naphthoic Acid by Flash Photolysis Experiments and Brownian Dynamics Simulations: Substantial Effects of Polarizability of the Triplet State.

Diffusional encounter rate constants, for xanthone and 2-naphthoic acid molecules in their triplet states with xanthone or 2-naphthoic acid molecules in their triplet or singlet states, were determined using nanosecond laser flash photolysis spectroscopy. Simultaneously, Brownian dynamics simulations were used to compute these rate constants for assumed models of encountering molecules. Altogether, a global fit to transient absorption progress curves, reporting populations of triplet state xanthone and triplet state 2-naphthoic acid molecules, allowed us to determine six diffusional encounter rate constants from our experiments. The most important result of this study is the detection of substantial effects of the electric polarizability of molecules in their triplet state, visible for xanthone triplet and 2-naphthoic acid ground states, a homo triplet-triplet annihilation of 2-naphthoic acid, and a hetero triplet-triplet annihilation for xanthone and 2-naphthoic acid.

Improvement of cognitive functions in response to a regular Nordic walking training in elderly women - A change dependent on the training experience.

Although regular physical activity is known to benefit health of aging populations, there are still many factors, which regulate exercise-induced adaptive changes. Among many vitamin D and myokines are under consideration. We, therefore, evaluated the influence of a single session of and regular Nordic Walking (NW) training combined with vitamin D supplementation on cognitive functions and muscle strength and some elements of the amino-acid profile. Thirty-five healthy elderly women (68 ±â€¯5 years old) from health promotion programmes took part in the study. At baseline they were divided into two groups: women, who participated in NW training for the first time (Beginners Group: BG) and women, who continued regular NW training longer than four years (Advance Group: AG). All women had a similar concentration of vitamin D (above 20 ng·ml-1) at baseline. The 12 weeks of NW training was supported by supplementation of vitamin D3 (4000 IU/day). Muscle strength, serum concentrations of myokines (irisin and IL-6), brain derived neurotrophic factor (BDNF), inflammation marker, glucose, branched amino acids and tryptophan were all assessed at baseline, 1 h after the first single training session and adequately at the end of the training programme. In addition, iron and ferritin were measured. The concentration of vitamin D3 as well as psychological (Quality-of-Life Assessment, The Beck Depression Inventory-2) and cognitive evaluations (D2 test of attention, Trial Making Test A&B) were also performed before and after the 12-week training programme. Data were interpreted using magnitude-based inferences. According to data obtained in this study, regular NW training resulted in improvement of cognitive functions in aged women. These positive changes were accompanied by an increase of irisin and BDNF concentration (adjusted effect moderate and likely). Our data also revealed that observed reductions of glucose and tryptophan concentrations might have positively contributed to the amelioration of cognitive functions. Still, obtained results indicated that it was not the level vitamin D that modulated exercise-induced changes, but rather the long-lasting experience and being more advanced in training.

Kinetics of binding the mRNA cap analogues to the translation initiation factor eIF4E under second-order reaction conditions.

The kinetics of binding of five analogues of the 5'-mRNA cap, differing in size and electric charge, to the eukaryotic initiation factor eIF4E, at 20 degrees C, pH 7.2, and ionic strength of 150 mM, were measured, after mixing solutions of comparable concentrations of the reagents, in a stopped-flow spectrofluorimeter. The registered stopped-flow signals were fitted using an efficient software package, called Dyna Fit, based on a numerical solution of the kinetic rate equations for assumed reaction mechanisms. One-, two-, and three-step binding models were considered. The quality of fits for these models were compared using two statistical criteria: Akaike's Information Criterion and Bayesian Information Criterion. Based on resulting probabilities of the models, it was concluded that for all investigated ligands a one-step binding model has essentially no support in the experimental observations. Our conclusions were also analysed from the perspective of kinetic transients obtained for cap-eIF4E systems under the so called pseudo-first order reaction condition, which result in the linear correlation of the observed association rate constant with ligand concentration. The existence of such a linear correlation is usually considered as proof of a one-step binding mechanism. The kinetic and optical parameters, derived from fitting a two-step cap-binding model with the DynaFit, were used to simulate kinetic transients under pseudo-first order reaction conditions. It appeared that the observed association rate constants derived from these simulated transients are also linearly correlated with the ligand concentration. This indicated that these linear dependencies are not sufficient to conclude a one-step binding.

Towards the mechanism of trimeric purine nucleoside phosphorylases: stopped-flow studies of binding of multisubstrate analogue inhibitor - 2-amino-9-[2-(phosphonomethoxy)ethyl]-6-sulfanylpurine.

The binding of multisubstrate analogue inhibitor - 2-amino-9-[2-(phosphonomethoxy)ethyl]-6-sulfanylpurine (PME-6-thio-Gua) to purine nucleoside phosphorylase from Cellulomonas sp. at 20 degrees C, in 20 mM Hepes buffer with ionic strength adjusted to 50 mM using KCl, at several pH values between 6.5 and 8.2, was investigated using a stopped-flow spectrofluorimeter. The kinetic transients registered after mixing a protein solution with ligand solutions of different concentrations were simultaneously fitted by several association reaction models using nonlinear least-squares procedure based on numerical integration of the chemical kinetic equations appropriate for given model. It is concluded that binding of a PME-6-thio-Gua molecule by each of the binding sites is sufficiently well described by one-step process, with a model assuming interacting binding sites being more probable than a model assuming independent sites. The association rate constants derived from experimental data, assuming one step binding and independent sites, are decreasing with an increase in pH, changing from 30 to 6 microM(-1)s(-1) per binding site. The dissociation rate constants are in the range of 1-3 s(-1), and they are rather insensitive of changes in pH. Interestingly, for each pH value, the one-step binding model with interacting sites results in the association rate constant per site 1.5-4 times smaller for the binding of the first ligand molecule than that for the binding of the second one. Decrease of association constants with pH indicate that the enzyme does not prefer binding of the naturally occurring anionic form of the 6-thioguanine ring (pK(a) 8.7) resulting from a dissociation of N(1)-H. This finding supports the mechanism in which hydrogen bond interaction of N(1)-H with Glu204 (Glu 201 in mammalian PNPs) is crucial in the catalytic process. Results obtained also indicate that, in contrast to transition-state analogues, for which binding is followed by a conformational change, binding of multisubstrate analogue inhibitors to trimeric PNPs is a one-step process.

A common oxygen sensor regulates the sensory discharge and glomus cell HIF-1alpha in the rat carotid body.

The relationships between the chemosensory discharge and glomus cells HIF-1alpha (hypoxia inducible factor 1alpha) in the rat carotid body in vitro were investigated using CO as a tool. Both chemosensory discharge and HIF-1alpha were stimulated by CO, although the former took only a few seconds and the latter a few minutes to develop. These developments were suppressed by light. By using the monochromatic lights, the action spectra were prepared. Lights of 430 nm 590 nm were most effective in suppressing the responses. It is known that these lights are the signature markers for cytochrome oxidase, making identification of the entities. Thus, cytochrome oxidase serves as a common oxygen sensor for both.

Myokines in Response to a Tournament Season among Young Tennis Players.

The study investigated changes in myokines, heat shock proteins, and growth factors in highly ranked, young, male tennis players in response to physical workload during the competitive season and their potential correlations with match scores. Blood collections were carried out at the beginning, the midpoint, and the end of the tournament season. Data analysis revealed a significant increase in interleukin 6 and its inverse correlation with the number of lost games (r = -0.45; 90% CI -0.06 to 0.77). Neither the irisin nor BDNF level changed notably, yet delta changes of irisin across the season significantly correlated with the number of games won. The concentration of HSP27 recorded a small increase (31.2%; 90% CI 10.7 to 55.5, most likely). A negative correlation was noted between IGF-1 and HSP27 concentration at baseline (-0.70 very high; 90% CI -0.89 to -0.31, very likely). At the end of the season IGF-1 correlated positively with the number of games won (r = 0.37 moderate, 90% CI -0.16 to 0.73, likely) but negatively with the number of games lost (r = -0.39, 90% CI -0.14 to -0.74, likely). In conclusion our data indicated that Il-6, irisin, and growth factor IGF-1 may modify overall performance during a long lasting season, expressed in the amount of games won or lost.

A new fluorescence method to detect singlet oxygen inside phospholipid model membranes.

A fluorescence method for detecting singlet oxygen (1O2) in model membranes is proposed. 1O2 was generated by hydrogen peroxide/sodium hypochlorite system. 1,3-Diphenylisobenzofuran (DPBF), a specific 1O2 trap, dissolved in organic solvents gives a strong fluorescence spectrum when excited at 410 nm. A similar spectrum, with a maximum at 455 nm, is obtained when DPBF is incorporated in unilamellar dipalmitoylphosphatidylcholine liposomes. The intensity of fluorescence spectrum decreases when DPBF-labeled liposomes are exposed to singlet oxygen. This decrease is sensitive to 1O2 traps and quenchers like tryptophan and sodium azide, to lipid membrane fluidity and to the concentration of sodium hypochlorite and hydrogen peroxide.

Prediction of pH-dependent properties of proteins.

We describe what may be the most accurate approach currently available for the calculation of the pKas of ionizable groups in proteins. The accuracy is assessed by comparison of computed pKas with 60 measured pKas in a total of seven proteins. The overall root-mean-square error is 0.89 pKa units. Linear regression analysis of computed versus measured pKas yields a slope of 0.95, y-intercept of -0.02 and a correlation coefficient of 0.96. The proposed approach also picks out many of the shifted pKas of groups in enzyme active sites and special salt bridges. However, it does yield several over-shifted pKas and tends to underestimate pKa shifts which result from desolvation effects. We examine the ability of the new approach to reproduce the dependence of protein stability upon pH, using the ionization polynomial formalism. Overall features of the stability curves are reproduced, but the quantitative agreement is not particularly good. The reasons for the disagreement may have to do both with insufficient accuracy in the theory and with uncertainty in the nature of the unfolded state of proteins. The methodology described here is based upon finite difference solutions of the Poisson-Boltzmann equation. Its success depends upon the use of the rather high protein dielectric constant of 20. However, theoretical considerations and the fact that pKa shifts which result from desolvation are underestimated here imply that the dielectric constant of the protein interior actually is lower than 20. We suggest that the high protein dielectric constant improves the overall agreement with experiment because it accounts approximately for phenomena which tend to mitigate pKa shifts and which are not specifically included in the model. These include conformational relaxation and specific ion-binding. Future models based upon a low protein dielectric constant and treating such phenomena explicitly might yield improved agreement with experiment.

Effects of solute-solvent proton exchange on polypeptide chain dynamics: a constant-pH molecular dynamics study.

A method for performing implicit-solvent molecular dynamics simulations at constant pH was applied to a pentapeptide acetyl-Ala-Asp-Ala-Lys-Ala-amide at pH 4. As a reference, molecular dynamics simulations were done for the same peptide with two variants of its fixed protonation patterns expected to dominate at pH 4, i.e., with a protonated and a deprotonated side chain of the Asp residue and the protonated Lys residue in both cases. The dynamic trajectories of the peptide were used to discuss the problem of the significance of the solute-solvent proton exchange phenomena for the dynamics and structural distributions of the polypeptide chain. The Asp-Lys distance was used as a probe of the overall molecular structure of the investigated pentapeptide. To characterize the dynamics, distributions of the "waiting" times for a transition from a "short" distance conformation to a "long" distance conformation were constructed, based on the generated molecular dynamics trajectories. We show that the relaxation time for the transitions, derived from the constant-pH simulations, is very close to the relaxation time characterizing a permanently protonated molecule, although the average protonation probability of the short-distance conformation is close to zero. However, the distribution of the Asp-Lys distances obtained from constant-pH simulations cannot be reproduced as a linear combination of the distributions resulting from the simulations with fixed protonation states.