Structural engineering of the HIV-1 protease molecule with a beta-turn mimic of fixed geometry.

An important goal in the de novo design of enzymes is the control of molecular geometry. To this end, an analog of the protease from human immunodeficiency virus 1 (HIV-1 protease) was prepared by total chemical synthesis, containing a constrained, nonpeptidic type II' beta-turn mimic of predetermined three-dimensional structure. The mimic beta-turn replaced residues Gly16,17 in each subunit of the homodimeric molecule. These residues constitute the central amino acids of two symmetry-related type I' beta-turns in the native, unliganded enzyme. The beta-turn mimic-containing enzyme analog was fully active, possessed the same substrate specificity as the Gly16,17-containing enzyme, and showed enhanced resistance to thermal inactivation. These results indicate that the precise geometry of the beta-turn at residues 15-18 in each subunit is not critical for activity, and that replacement of the native sequence with a rigid beta-turn mimic can lead to enhanced protein stability. Finally, the successful incorporation of a fixed element of secondary structure illustrates the potential of a "molecular kit set" approach to protein design and synthesis.     

Selection of synthetic proteins to modulate the human frataxin function

Frataxin is a kinetic activator of the mitochondrial supercomplex for iron-sulfur cluster assembly. Low frataxin expression or a decrease in its functionality results in Friedreich's Ataxia (FRDA). With the aim of creating new molecular tools to study this metabolic pathway, and ultimately, to explore new therapeutic strategies, we have investigated the possibility of obtaining small proteins exhibiting a high affinity for frataxin. In this study, we applied the ribosome display approach, using human frataxin as the target. We focused on Affi_224, one of the proteins that we were able to select after five rounds of selection. We have studied the interaction between both proteins and discussed some applications of this specific molecular tutor, concerning the modulation of the supercomplex activity. Affi_224 and frataxin showed a K_D value in the nanomolar range, as judged by surface plasmon resonance analysis. Most likely, it binds to the frataxin acidic ridge, as suggested by the analysis of chemical shift perturbations (nuclear magnetic resonance) and computational simulations. Affi_224 was able to increase Cys NFS1 desulfurase activation exerted by the FRDA frataxin variant G130V. Importantly, Affi_224 interacts with frataxin in a human cellular model. Our results suggest quaternary addition may be a new tool to modulate frataxin function in vivo. Nevertheless, more functional experiments under physiological conditions should be carried out to evaluate Affi_224 effectiveness in FRDA cell models.     

Correlation between protein stability and crystal properties of designed ROP variants

Six variants of the ROP protein, designed with the aim to analyze by X-ray crystallography loop formation and core packing interactions in 4-α-helical bundles- have been purified and a search of their crystallization conditions has been carried out. Five mutants yield crystals that are suitable for medium to high resolutionX-ray diffraction studies. For all mutants crystal size- sensitivity to X-irradiation and diffraction limit are correlated to their stability as determined by differential scanning calorimetry- in a manner which is not yet understood in detail. © Wiley-Liss, Inc.     

Population dynamics of japanese serow in relation to social organization and habitat conditions. I. Stability of Japanese serow density in stable habitat conditions

The density of a Japanese serow (Capricornis crispus) population, which has been in stable habitat conditions and free from hunting and predation, was investigated by direct observation of serows at Kusoudomari (336 ha), Wakinosawa village, Aomori Prefecture, Japan, during 1977–89. The vegetation, composed of mature mixed forest ofThujopsis with deciduous trees (43.2%), secondary forest of deciduous trees (23.4%) and a plantation of coniferous trees (30.0%) showed little change during the study period. The serows were counted 17 times each winter for 14 consecutive years. The serow population maintained a stable density with a mean of 12.5 individuals km⁻² (SD=1.4) ranging from 8.6 to 14.6 km⁻². The ratio of the proportion of kids (<1 year old) to serows more than 1 year old decreased, but density did not decrease during the study period. Though extremely severe winters with heavy snowfall in 1984–86 caused a decline in serow density, the density recovered within a few years. It was considered that the stability of the serow density resulted mainly from the stable food supply and the lack of human disturbance, such as forest cutting in the habitat. Stable population density for a long period is probably related to the social organization of the serow.     

STABILIZATION MECHANISM IN SWIMMING ODONTOCETE CETACEANS BY PHASED MOVEMENTS

Propulsive movements of the caudal oscillating flukes produce large forces that could induce equally large recoil forces at the cranial end of the animal, and, thus, affect stability. To examine these vertical oscillations, video analysis was used to measure the motions of the rostrum, pectoral flipper, caudal peduncle, and fluke tip for seven odontocete cetaceans: Delphinapterus leucas, Globicephala melaena, Lagenorhynchus obliquidens, Orcinus orca, Pseudorca crassidens, Stenella plagiodon , and Tursiops truncatus. Animals swam over a range of speeds of 1.4–7.30 m/sec. For each species, oscillatory frequency of the fluke tip increased linearly with swimming speed. Peak-to-peak amplitude at each body position remained constant with respect to swimming speed for all species. Mean peak-to-peak amplitude ranged from 0.02 to 0.06 body length at the rostrum and from 0.17 to 0.25 body length at the fluke tip. The phase relationships between the various body components remain constant with respect to swimming speed. Oscillations of the rostrum were nearly in phase with the fluke tip with phase differences out of—9.4°-33.0° of a cycle period of 360°. Pectoral flipper oscillations trailed fluke oscillations by 60.9°-123.4°. The lower range in amplitude at the rostrum compared to the fluke tip reflects increased resistance to vertical oscillation at the cranial end, which enhances the animal's stability. This resistance is likely due to both active and passive increased body stiffness, resistance on the flippers, phased movements of body components, and use of a lift-based propulsion. Collectively, these mechanisms stabilize the body of cetaceans during active swimming, which can reduce locomotor energy expenditure and reduce excessive motions of the head affecting sensory capabilities.     

Relationship between grass species richness and ecosystem stability in Kruger National Park, South Africa

The relationship between grass species richness and ecosystem stability was investigated in the Kruger National Park. A total of 135 489 individual grasses were identified from 189 sites spread across 19 485 km² of savanna biome, making this one of the largest studies of its kind. At each site, grass percentage abundance and standing crop were measured at one year intervals to provide an index of ecosystem function. A severe drought that affected the region between 1991 and 1993 provided a convenient perturbation. At the height of the drought, mean species richness declined by 12.7% while standing crop declined by 38.1%, from 3199 to 1979 kg ha^?1. Percentage grass abundance declined to 87.5% of its pre‐drought value. After the drought had passed species richness, standing crop and percentage abundance recovered to 92.1%, 113.8% and 92.8% of their pre‐perturbation values, respectively. Statistical analysis of these responses revealed that grass assemblages of low species richness were more resistant to drought than assemblages of high species richness. Species‐poor sites also showed better recovery from perturbation after the drought had passed. These findings suggest that ecosystem stability may be negatively related to grass species richness in South African savanna grasslands.     

Insights into the local residual entropy of proteins provided by NMR relaxation.

A simple model is used to illustrate the relationship between the dynamics measured by NMR relaxation methods and the local residual entropy of proteins. The expected local dynamic behavior of well-packed extended amino acid side chains are described by employing a one-dimensional vibrator that encapsulates both the spatial and temporal character of the motion. This model is then related to entropy and to the generalized order parameter of the popular "model-free" treatment often used in the analysis of NMR relaxation data. Simulations indicate that order parameters observed for the methyl symmetry axes in, for example, human ubiquitin correspond to significant local entropies. These observations have obvious significance for the issue of the physical basis of protein structure, dynamics, and stability.