Application of tyrosine-tryptophan fluorescence resonance energy transfer in monitoring protein size changes.

Monitoring protein size changes has versatile applications in studying protein folding/unfolding, conformational rearrangements, and ligand binding. Traditionally, FRET has been used to obtain this information. However, the use of FRET often requires covalent attachment of exogenous fluorophores. Although intrinsic FRET also exists between tyrosine and tryptophan residues, it has been underused because of tyrosinate formation and spectroscopic overlap. Herein, we clarified the concern of tyrosinate formation and mathematically deconvoluted tyrosine/tryptophan fluorescence spectra. We define a new parameter called FirbY-W (fluorescence intensity ratio between tyrosine and tryptophan) to reflect protein sizes. We demonstrate its applications in studying protein unfolding using several model proteins. In all the cases, our method offers superior sensitivity, data quality, and robustness compared with traditional techniques. The unique power of our method is in its ability to detect elusive conformational changes of intrinsically disordered proteins (IDP). The lack of structure makes IDPs unsuitable for CD or tryptophan fluorescence characterization. Using histone mRNA stem-loop binding protein (SLBP) as an example of disordered proteins, we showed that our method is capable of detecting conformational changes caused by phosphorylation, which are effectively invisible for traditional spectroscopic methods. Our method can also be used to detect RNA binding of disordered proteins.

Effect of fasting on body composition and responses to stress in sunshine bass.

The integrated responses of the hormonal regulation of growth and stress in sunshine bass (Morone chrysops X Morone saxatilis) as regulated by feed deprivation were investigated. Groups of fish were fed 1.5% of the body weight per day or offered no feed for 4weeks. Another group of fish was not fed for 3weeks and feed was offered during the fourth week. Fish in each group were sampled immediately before or after a 15-min low water confinement stressor after each week of the experiment. Liver mass and liver glycogen content were decreased after one week of fasting and remained low until the end of the study. However, both recovered after a week of refeeding. Intraperitoneal fat was significantly lower after two weeks of fasting and did not recover after a week of refeeding. None of these components were affected by confinement stress. Plasma glucose in unstressed fish was generally unaffected by fasting or refeeding; however, plasma glucose increased after confinement stress in fed but not in fasted fish. The cortisol stress response was unaltered by fasting and remained robust. Plasma IGF-I generally decreased in fasted fish but was not significantly lower than fed fish until the fourth week. A week of refeeding did not restore plasma IGF-I concentrations. Plasma IGF-I concentrations were higher in confinement stressed fed fish after two and four weeks but were unchanged in the fourth week. There was no change in the plasma IGF-I concentrations in fasted or refed fish due to the stress. Liver weight and liver glycogen were essentially depleted after 2weeks of fasting. The reduction of liver glycogen greatly reduced the glucose response to stress; however, the cortisol stress response was maintained for at least four weeks of fasting. Intraperitoneal fat was decreased very little after 4weeks of fasting. Plasma IGF-I concentrations were reduced only after 3weeks of fasting.

Influence of reproductive status, sex hormones and temperature on plasma IGF-I concentrations in sunshine bass (Morone chrysops x Morone saxatilis).

Insulin-like growth factor-I (IGF-I) concentrations in male and female sunshine bass (Morone chrysops x Morone saxatilis) were determined in March, early April, and late April in outdoor ponds at a commercial farm. Female fish were always larger than male fish; however, plasma IGF-I concentrations tended to be higher in male fish and increased as pond temperature and feeding increased in both sexes. Gonadal development was greatest in both sexes in March and declined to a regressed state by the end of April and the same pattern of change occurred with plasma estrogen and testosterone. Growth and IGF-I concentrations in sunshine bass fed estrogen, methyl testosterone, or a control diet were also determined. Growth was reduced in fish fed both sex hormones. Fish fed the control diet had the highest IGF-I levels, androgen-fed fish had intermediate levels, and estrogen-fed fish had the lowest IGF-I concentrations after 4 weeks on the diet. Plasma IGF-I concentrations appeared to respond to increasing temperature in the ponds, and were inversely related to gonadal development and sex hormones. Exogenous sex hormones resulted in a decrease in plasma IGF-I, feeding activity and growth.

The effect of temperature, stress, and cortisol on plasma IGF-I and IGFBPs in sunshine bass.

The mechanisms through which stress and cortisol regulate insulin like growth factor-I (IGF-I) and insulin like growth factor binding proteins (IGFBPs) were studied in sunshine bass, by measuring plasma IGF-I and IGFBPs in fish maintained at 5, 10, 15, 20, 25, or 30 degrees C, fish subjected to an acute 15 min confinement stress at 25 and 30 degrees C, and fish fed 100 mg cortisol/kg feed. Plasma IGF-I concentrations were higher at 25 and 30 degrees C than at 20 degrees C and below. A 15 min confinement stress resulted in a decrease in IGF-I 2h post-confinement. Plasma concentrations of IGFBP with molecular weights of 24, 28, and 33 kDa were similar for fish acclimated to different temperatures, except for 5 degrees C where a 33-kDa IGFBP was significantly reduced. After a 15 min low-water stress at 25 degrees C, a 33-kDa IGFBP was reduced and IGFBPs with molecular weights of 24 and 28 kDa were increased at 2 and 6h, respectively. A 15 min low-water stress at 30 degrees C, resulted in no change in levels of a 33-kDa IGFBP over the 6-h recovery period. However, levels of a 24- and 28-kDa IGFBP were significantly increased at 2 and 6h, respectively. A single feeding with 100 mg cortisol/kg feed increased plasma cortisol but did affect plasma concentrations of IGF-I or any of the three IGFBPs. Acute stress appears to result in a decrease in IGF-I, but the mechanism of the decrease does not appear to be caused by cortisol released during the stress.

Rates of cortisol increase and decrease in channel catfish and sunshine bass exposed to an acute confinement stressor.

Channel catfish and sunshine bass were exposed to a low-water stress event and allowed to recover in fresh water or a solution of metomidate (dl-1-(1-phenylethyl)-5-(metoxycarbonyl) imidazole hydrochloride), which inhibits the synthesis of cortisol. Change in time of plasma cortisol was used as an index of cortisol secretion and clearance. Plasma cortisol and glucose increased during the exposure to low-water stress in both fish, but the changes of both plasma components were more dramatic in sunshine bass. Exposure to metomidate during recovery resulted in a short-term increase in plasma glucose but differences between controls and metomidate-exposed fish were relatively minor thereafter. Cortisol began to decrease in catfish immediately after the removal of the stress but continued to increase for 15 min in sunshine bass recovering in fresh water and for 5 min in bass recovering in metomidate. Catfish recovering in fresh water had a cortisol elimination rate of -1.28 ng/mL/min compared with -2.45 ng/mL/min for fish recovering in metomidate (P>0.05) while sunshine bass recovering in fresh water had an elimination rate of -6.96 ng/mL/min compared with -4.50 ng/mL/min for fish recovering in metomidate (P>0.05). These data indicate that the rapid decrease of plasma cortisol after removal of the stressor is due to an almost immediate decrease of secretion, tissue uptake and a rapid renal loss due to the absence of a plasma binding protein.

Temperature affects physiological stress responses to acute confinement in sunshine bass (Morone chrysops x Morone saxatilis).

Sunshine bass (Morone chrysopsxMorone saxatilis) were subjected to a 15-min low-water confinement stressor at temperatures ranging from 5 to 30 degrees C. Physiological responses were evaluated by measuring hematocrit, and plasma chloride, glucose and cortisol. Fish acclimated to 30 degrees C had initial glucose concentrations of 3.13 mM (564 mg/L) which were significantly lower than in fish acclimated to 5 and 10 degrees C (4.32 and 4.82 mM or 779 and 868 mg/l, respectively). Fish survived the conditions imposed at every temperature except 30 degrees C, where 15 out of 42 fish died during the stress and recovery protocol. The general pattern was an initial increase in hematocrit, followed by a delayed decrease in hematocrit and chloride, and an increase in plasma glucose and cortisol. In general, fish stressed at temperatures below 20 degrees C had lower and more delayed changes in plasma glucose and cortisol than fish tested at 20, 25 and 30 degrees C. Initial cortisol concentrations were 65 ng/ml and increased to above 200 ng/ml in fish held at 20 degrees C and above. At the higher temperatures, glucose concentrations were twice the initial concentration after stress and cortisol changes were four to five times the initial concentration after the stress. Quantitative responses for glucose and cortisol were moderate and recovery rapid in fish stressed at 10 and 15 degrees C; therefore, this range of water temperature is recommended when handling sunshine bass.