Lactam bridge stabilization of α-helical peptides: Ring size,orientation and positional effects

A series of 14 residue amphipathic α-helical peptides, in which the sidechains of glutamic acid and lysine have been covalently joined, was synthesized in order to determine the effect of spacing, position and orientation of these lactam bridges. It was found that although an (i, i+3) spacing would position the lactam bridge on the same face of the helix, these lactams with 18-member rings were actually helix-destabilizing regardless of position or location. On the other hand, (i, i+4) lactams with 21-member rings were helix-stabilizing but this was dependent on orientation. Glutamic acid-lysine lactams increased the helical content of the peptide when compared with their linear homologue in benign conditions (50 mM KH₂PO₄, 100 mM KCl, pH 7). Two Glu-Lys (i, i+4) lactams located at the N- and C-termini gave rise to a peptide with greater than 99% helical content in benign conditions. Peptides with Lys-Glu oriented lactams were random structures in benign conditions but in the presence of 50% TFE could be induced into a helical conformation. The stability of the single-stranded α-helices, as measured by thermal denaturations in 25% TFE indicated that Glu-Lys oriented lactam bridges stabilized the helical conformation relative to the linear unbridged peptide. One Glu-Lys lactam in the middle of the peptide was more effective at stabilizing helical structure than two Glu-Lys lactams positioned one at each end of the molecule. The lactams with the Lys-Glu orientation were destabilizing relative to the unbridged peptide. This study demonstrates that correct orientation and position of a lactam bridge is critical in order to design peptides with high helical content in aqueous media.     

Avian daily foraging patterns: Effects of digestive constraints and variability

Summary Birds show a typical daily pattern of heavy morning and secondary afternoon feeding. We investigate the pattern of foraging by a bird that results in the lowest long-term rate of mortality. We assume the following: mortality is the sum of starvation and predation. The bird is characterized by two state variables, its energy reserves and the amount of food in its stomach. Starvation occurs during the day if the bird's reserves fall to zero. The bird starves during the night if the total energy stored in reserves and the stomach is less than a critical amount. The probability that the bird is killed by a predator is higher if the bird is foraging than if it is resting. Furthermore, the predation risk while foraging increases with the bird's mass. From these assumptions, we use dynamic programming techniques to find the daily foraging routine that minimizes mortality. The principal results are (1) Variability in food finding leads to routines with feeding concentrated early in the day, (2) digestive constraints cause feeding to be spread more evenly through the day, (3) even under fairly severe digestive constraints, the stomach is generally not full and (4) optimal fat reserve levels are higher in more variable environments and under digestive constraints. This model suggests that the characteristic daily feeding pattern of small birds is not due to digestive constraints but is greatly influenced by environmental variability.     

Variation in heat-shock proteins among species of desert fishes (Poeciliidae, Poeciliopsis)

Analysis of the heat-shock proteins (hsps) of six closely related species of Poeciliopsis demonstrated the existence of biochemical diversity in the hsp100, hsp70, hsp60, and hsp30 protein families among species. Each species expressed five to seven hsp70-related isoforms. Constitutive 70-kD isoforms were identical among species, but four different patterns of heat-inducible isoforms were seen in these six species. Members of the hsp70 family of molecular chaperones are included among the most highly conserved proteins known, and the possibility of variation in hsp70 among closely related species has rarely been addressed. The hsp30 family is known to be less conserved than the hsp70 family, and, as expected, the Poeciliopsis hsp30 patterns showed more variation. Most of the hsp30 isoforms characteristic of a particular species were unique to that species. Hsp100 and hsp60 were identical in five of the species, but alternate isoforms were found in P. monacha. The small size and limited geographical distribution of the P. monacha population have probably contributed to the uniqueness of the monacha pattern. Two of the species were shown to acquire thermotolerance, the ability to withstand normally lethal temperatures when subjected to a gradual temperature increase. Rapid-heating protocols commonly used to establish critical thermal maxima of organisms do not include this inducible component of thermoresistance and therefore do not adequately assess an organism's capacity to withstand thermal stress.      

The Value of a hoard: not just energy

We present a stochastic dynamic programming model of the contributionof stored food to winter survival. Using the acorn woodpecker(Melanerpes formtcivorus) as a model organism, we demonstratethat a hoard of small energetic value can impart considerableimprovement in the probability of surviving the winter and soimprove fitness. With this model we hope to resolve Koenig andMumme's paradox, that acorn woodpeckers expend much time andeffort to create and maintain hoards of acorns which provideno more that 16% of their energetic needs over the period inwhich they are used. We further demonstrate that the contributionof hoarded acorns to survival depends on the variability inforaging outcome, independently of the energetic value of thehoard in absolute terms. We point out that the results applyin principle to all hoarding animals and suggest a number offurther elaborations of the model.     

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.      

Arming α2-macroglobulin with ricin A chain forms a cytotoxic conjugate that inhibits protein synthesis and kills human fibroblasts

A conjugate containing α₂-macroglobulin and highly purified ricin A chain was made using N-succinimidyl-3-(2-pyridyldithio)propionate. Radioimmunoassay indicated that it contained 1.2 mol A chain per mol α₂-macroglobulin. The conjugate inhibited polyuridylic-acid directed translation by rat liver ribosomes and protein synthesis in human fibroblasts. There was a 90 min lag period before the beginning of inhibition in fibroblasts, but complete inhibition could be achieved. By measuring protein synthesis as a function of protein concentration, it was demonstrated that 8.25·10^?9M conjugate was required to inhibit 50% of protein synthesis in 6 h. To achieve the same level of inhibition, 165-times more (1.3·10^?6M) unconjugated A chain was required, and 180-times less ricin (4.6·10^?11M). Ricin was more than 28 000 times more inhibitory than A chain alone. The presence of α₂-macroglobulin did not increase the cytotoxicity of unconjugated A chain, and it even protected the cells to a slight extent. The inhibitory action of the conjugate was blocked by antibodies specific for α₂-macroglobulin or ricin, and it was not prevented by galactose or antibodies specific for ricin B chain. Electron microscopy of the conjugate indirectly labelled with ferritin demonstrated that it was internalized by receptor mediated endocytosis through coated pits. These data indicate that the A chain portion of the conjugate survives the conditions in the lysosomes to the extent that it retains its ability to inactivate cytoplasmic ribosomes.     

Torque potentiation and myosin light-chain phosphorylation in human muscle following a fatiguing contraction

Indices of electrically stimulated and maximal voluntary isometric muscle torgue and the phosphate content of myosin phosphorylatable light chains (P light chains) were studied during recovery following a 60-s maximal voluntary isometric contraction (MVC) in 21 human subjects. Analysis of muscle biopsy samples revealed that immediately after the 60-s MVC there were significant decreases in ATP (-15%) and phosphocreatine (-82%), and lactate concentration increased by 17-fold. All indices of muscle torque production were reduced by the 60-s MVC, but the twitch torque and torque at 10 Hz were relatively less reduced compared with the torque at 20 and 50 Hz or a 1-s MVC. Between 3 and 6 min of recovery, twitch torque and torque at 10 Hz stimulation were significantly potentiated, reaching peak values of 125 and 134%, respectively, compared with rest. Phosphate content of the fast and two slow P light chains was significantly increased over rest levels immediately after and 4 min after the 60-s MVC. These results suggest that myosin P light-chain phosphorylation could provide a mechanism to increase human muscle torque under conditions of submaximal contractile element activation following fatigue.