Quantitative analysis of the composition of the native and scrambled ribonuclease A

Reversible conversion between the native and scrambled proteins can be applied to analyze the denaturation curve of a disulfide-containing protein. In the case of RNase A, scrambled species could not be well separated from the native species by HPLC to permit precise quantitative analysis of the extent of denaturation. Methods are developed here to overcome this problem. The methods exploit the difference of conformational stability between the native and scrambled RNase A. When a sample of partially denatured RNase A was placed under mild reducing conditions (0.2-1 mM dithiothreitol for 10 min), the disulfide bonds of the native RNase A remain intact, whereas those of scrambled isomers become fully reduced. The native and fully reduced species of RNase A can be completely separated by HPLC. Alternatively, a mixture of partially denatured RNase A can be treated with mild concentration of proteolytic enzymes (trypsin or thermolysin). In this approach, scrambled isomers of RNase A were totally fragmented and readily separated from the native RNase A. These methods allow analysis and construction of the denaturation curves of RNase A in the presence of urea, GdmCl and GdmSCN.     

Future directions in folding: The multi-state nature of protein structure

All possible protein folding intermediates exist in equilibrium with the native protein at native as well as non-native conditions, with occupation determined by their free energy level. The study of these forms can illuminate the fundamental principles of protein structure and folding. Hydrogen exchange methods can be used to detect and characterize these partially unfolded forms at native conditions and as a function of mild denaturant and temperature. This information illuminates the requirements that govern the ability of kinetic and equilibrium methods to study folding intermediates.     

Towards more robust methods of alien gene detection

Because the properties of horizontally-transferred genes will reflect the mutational proclivities of their donor genomes, they often show atypical compositional properties relative to native genes. Parametric methods use these discrepancies to identify bacterial genes recently acquired by horizontal transfer. However, compositional patterns of native genes vary stochastically, leaving no clear boundary between typical and atypical genes. As a result, while strongly atypical genes are readily identified as alien, genes of ambiguous character are poorly classified when a single threshold separates typical and atypical genes. This limitation affects all parametric methods that examine genes independently, and escaping it requires the use of additional genomic information. We propose that the performance of all parametric methods can be improved by using a multiple-threshold approach. First, strongly atypical alien genes and strongly typical native genes would be identified using conservative thresholds. Genes with ambiguous compositional features would then be classified by examining gene context, including the class (native or alien) of flanking genes. By including additional genomic information in a multiple-threshold framework, we observed a remarkable improvement in the performance of several popular, but algorithmically distinct, methods for alien gene detection.     

Observing single biomolecules at work with the atomic force microscope

Progress in the application of the atomic force microscope (AFM) to imaging and manipulating biomolecules is the result of improved instrumentation, sample preparation methods and image acquisition conditions. Biological membranes can be imaged in their native state at a lateral resolution of 0.5-1 nm and a vertical resolution of 0. 1-0.2 nm. Conformational changes that are related to functions can be resolved to a similar resolution, complementing atomic structure data acquired by other methods. The unique capability of the AFM to directly observe single proteins in their native environments provides insights into the interactions of proteins that form functional assemblies. In addition, single molecule force spectroscopy combined with single molecule imaging provides unprecedented possibilities for analyzing intramolecular and intermolecular forces. This review discusses recent examples that illustrate the power of AFM.     

Automated search of natively folded protein fragments for high-throughput structure determination in structural genomics

Structural genomic projects envision almost routine protein structure determinations, which are currently imaginable only for small proteins with molecular weights below 25,000 Da. For larger proteins, structural insight can be obtained by breaking them into small segments of amino acid sequences that can fold into native structures, even when isolated from the rest of the protein. Such segments are autonomously folding units (AFU) and have sizes suitable for fast structural analyses. Here, we propose to expand an intuitive procedure often employed for identifying biologically important domains to an automatic method for detecting putative folded protein fragments. The procedure is based on the recognition that large proteins can be regarded as a combination of independent domains conserved among diverse organisms. We thus have developed a program that reorganizes the output of BLAST searches and detects regions with a large number of similar sequences. To automate the detection process, it is reduced to a simple geometrical problem of recognizing rectangular shaped elevations in a graph that plots the number of similar sequences at each residue of a query sequence. We used our program to quantitatively corroborate the premise that segments with conserved sequences correspond to domains that fold into native structures. We applied our program to a test data set composed of 99 amino acid sequences containing 150 segments with structures listed in the Protein Data Bank, and thus known to fold into native structures. Overall, the fragments identified by our program have an almost 50% probability of forming a native structure, and comparable results are observed with sequences containing domain linkers classified in SCOP. Furthermore, we verified that our program identifies AFU in libraries from various organisms, and we found a significant number of AFU candidates for structural analysis, covering an estimated 5 to 20% of the genomic databases. Altogether, these results argue that methods based on sequence similarity can be useful for dissecting large proteins into small autonomously folding domains, and such methods may provide an efficient support to structural genomics projects.     

Means for the assessment of radioligand quality and its importance in receptor-binding studies. Observations with radiolabelled formylmethionyl-leucyl-phenylalanine.

Various methods for testing the quality of radioligands were applied to two different radiolabelled forms of formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). The purpose of the study was both to examine the value of these methods for assessing radioligand quality and to determine the suitability of these particular radioligands for studying the chemotactic formylpeptide receptors on the rabbit neutrophil. It is useful in this context to distinguish two different aspects of radioligand quality: these are purity and equivalence to the native ligand. The two methods described for measuring receptor-reactivity (or 'bindability'), by measuring binding to an increasing excess of receptors and by a re-incubation procedure, provide a reliable measure of purity that should readily be applicable to other radioligands. Equivalence to the native ligand is more difficult to establish, and any uncertainty about the specific radioactivity of the radioligand can pose serious problems with this assessment. Commercial preparations of both tritiated and 35S-labelled fMet-Leu-Phe were found to be inadequately pure for detailed receptor studies. Repurification by t.l.c., however, consistently yielded radioligand preparations of high purity and close equivalence to the native ligand. Other radioligands may often also require a suitable repurification step before use for detailed receptor studies; this is especially important whenever a complex receptor-binding pattern is envisaged.     

Molecular Markers Allow to Remove Introgressed Genetic Background: A Simulation Study

The maintenance of genetically differentiated populations can be important for several reasons (whether for wild species or domestic breeds of economic interest). When those populations are introgressed by foreign individuals, methods to eliminate the exogenous alleles can be implemented to recover the native genetic background. This study used computer simulations to explore the usefulness of several molecular based diagnostic approaches to recover of a native population after suffering an introgression event where some exogenous alleles were admixed for a few generations. To remove the exogenous alleles, different types of molecular markers were used in order to decide which of the available individuals contributed descendants to next generation and their number of offspring. Recovery was most efficient using diagnostic markers (i.e., with private alleles) and least efficient when using alleles present in both native and exogenous populations at different frequencies. The increased inbreeding was a side-effect of the management strategy. Both values (% of native alleles and inbreeding) were largely dependent on the amount of exogenous individuals entering the population and the number of generations of admixture that occurred prior to management.     

Density-dependent grazing impacts of introduced European rabbits and sympatric kangaroos on Australian native pastures

Little information is available on relationships between pest animal density and damage in natural ecosystems. Introduced European rabbits, Oryctolagus cuniculus, cause severe damage to Australian native vegetation but density–damage relationships are largely unexplored. There are no recognized simple methods to estimate their impacts on native pastures, due in part to confusion with grazing impact of other herbivores. We tested simple quantitative sampling methods using multiple small quadrats to detect site differences in pasture cover, pasture species richness and dung pellet density of herbivores, from which rabbit density and relative abundance of larger herbivores were estimated. Native pasture cover and species richness declined exponentially with increasing rabbit density, within the range of 0–5 rabbits ha^?1, while cover of unpalatable exotic pasture species increased. By contrast, kangaroo abundance was positively related to palatable native pasture cover and negatively related to cover of unpalatable weeds, and had no negative effect on native pasture cover or species richness that was discernable against a background of low to moderate rabbit densities. Perennial native forbs and perennial grasses replaced invasive Wards weed as the dominant ground cover at low rabbit densities. We conclude that, regardless of previous grazing history, contemporary kangaroo grazing pressure and weed invasion, the severely degraded state of native pastures was perpetuated by rabbits. The effect of rabbits on native pasture can be recorded in a simple manner that is suitable for identifying density–damage relationships in the presence of other herbivores and changes over time. This method is seen as particularly useful in setting target densities below which rabbits must be managed to maintain native plant communities and ecosystem function in southern Australia. It may also be useful to demonstrate rabbits’ impacts in other regions, including optimum densities for plant biodiversity benefits in their native European range.     

Dissecting the roles of individual interactions in protein stability: lessons from a circularized protein

A circular form of bovine pancreatic trypsin inhibitor (BPTI) has been prepared by introducing a peptide bond between the N- and C-termini, which are in close proximity in the native conformation. The pathway and energetics of the disulphide-coupled folding transition of the circular protein have been studied using methods applied previously to the unmodified protein. The cross-link between the termini was found not to significantly stabilize the native state in spite of the expected reduction in entropy of the unfolded protein. This unexpected result has led to a reexamination of the stabilization expected from a cross-link, considering effects on the native, as well as unfolded, states of the protein. The greatest stabilization is expected when the cross-linked groups are held rigidly in the native protein in the optimum orientation for forming the cross-link. Similar analyses, utilizing thermodynamic cycles, can be applied to other interactions that stabilize native proteins, including disulphide bonds, salt bridges, and hydrogen bonds and to modifications to the protein that remove them. In general, the contribution of an individual interaction to the stability of the native state depends on the extent to which the interaction is favored in the native conformation, which can vary greatly depending on the local environment of the interacting groups.     

Comparison of activity and conformation changes during refolding of urea-denatured creatine kinase

The course of the recovery of the enzymatic activity and the native conformation during the renaturation of urea-denatured creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) has been studied. Under suitable conditions, an activity recovery of 95% can be obtained and the reactivation follows a triphasic course. The initial two phases are relatively fast, whereas the slow phase takes some 24 h to reach completion. The recovery of the native conformation has been followed by changes in fluorescence, ultraviolet absorption and in exposed SH groups and has been shown to be a biphasic process. Both the reactivation and the refolding processes are independent of protein concentrations within a certain range, showing that the dimerization of the enzyme molecule is not rate-limiting. A comparison of the rate constants for the refolding of the molecule with those for the recovery of its catalytic activity shows that these are not synchronized and the activity recovery approaches completion after the refolding and dimerization of the subunits so far as can be detected by the methods employed. The final stage of refolding with complete activity recovery probably involves subtle conformational changes of the dimeric enzyme molecule not detectable by the physiochemical methods used in the present study.     

Stable isotope and molecular analyses indicate that hybridization with non‐native domesticated common carp influence habitat use of native carp

Hybridization between native and non‐native species has consequences for survival and growth rates of hybrid offspring, but the influences on their functional roles such as habitat use are little studied and poorly understood. The Japanese native common carp Cyprinus carpio coexist and hybridize with non‐native domesticated carp in natural Japanese lakes. We have combined stable isotope and molecular information to examine whether habitat use of carp varies depending on the degree of hybridization between native and non‐native carp. We sampled 69 carp from Lake Kasumigaura where hybrid swarms between native and non‐native carp are advancing, evaluated the degree of hybridization for each individual by genotyping five single nucleotide polymorphism (SNP) markers, and analyzed their carbon and nitrogen stable isotopes. Although we did not find any genetically pure native carp in the lake, the results showed that carp δ¹³C increased with increasing frequency of non‐native alleles but that δ¹⁵N did not change. This indicates that non‐native carp use the littoral zone more frequently than native carp. This difference in habitat use was supported by a multisource mixing model, showing that the contribution of limnetic primary consumers to the diets of non‐native carp was lower than that of individuals with the highest frequency of native alleles. By combining two very different methods, our results thus suggest that multiple‐generation hybridization can influence habitat and resource use. Habitat partitioning should be considered when evaluating the genetic impacts of invasive species and races on native species and ecosystem processes.     

Ensuring seed quality in ecological restoration: native seed cleaning and testing

Seeds are a critical and limited resource for restoring biodiversity and ecological function to degraded and fragmented ecosystems. Cleaning and quality testing are two key steps in the native seed supply chain. Optimizing the practices used in these steps can ensure seed quality. Post‐collection handling of seeds can have a profound impact on their viability, longevity in storage, and establishment potential. The first section of this article describes seed cleaning, outlines key considerations, and details traditional and novel approaches. Despite the growth of the native seed industry and the need for seed quality standards, existing equipment and standards largely target agricultural, horticultural, and commercial forestry species. Native plant species typically have complex seed traits, making it difficult to directly transfer existing cleaning and quality standards to these species. Furthermore, in ecological restoration projects, where diversity is valued over uniformity crop standards can be unsuitable. We provide an overview and recommendations for seed quality testing (sampling, purity, viability, germinability, vigor), identity reporting, and seed transfer as well as highlight the need to implement internationally recognized standards for certification for native seeds. Novel and improved cleaning and testing methods are needed for native species from a range of ecosystems to meet the challenges and goals of the United Nations Decade on Ecosystem Restoration. The guidelines outlined in this article along with others in the Special Issue of Restoration Ecology “Standards for Native Seeds in Ecological Restoration” can serve as a foundation for this critical work.     

Modeling disordered regions in proteins using Rosetta

Protein structure prediction methods such as Rosetta search for the lowest energy conformation of the polypeptide chain. However, the experimentally observed native state is at a minimum of the free energy, rather than the energy. The neglect of the missing configurational entropy contribution to the free energy can be partially justified by the assumption that the entropies of alternative folded states, while very much less than unfolded states, are not too different from one another, and hence can be to a first approximation neglected when searching for the lowest free energy state. The shortcomings of current structure prediction methods may be due in part to the breakdown of this assumption. Particularly problematic are proteins with significant disordered regions which do not populate single low energy conformations even in the native state. We describe two approaches within the Rosetta structure modeling methodology for treating such regions. The first does not require advance knowledge of the regions likely to be disordered; instead these are identified by minimizing a simple free energy function used previously to model protein folding landscapes and transition states. In this model, residues can be either completely ordered or completely disordered; they are considered disordered if the gain in entropy outweighs the loss of favorable energetic interactions with the rest of the protein chain. The second approach requires identification in advance of the disordered regions either from sequence alone using for example the DISOPRED server or from experimental data such as NMR chemical shifts. During Rosetta structure prediction calculations the disordered regions make only unfavorable repulsive contributions to the total energy. We find that the second approach has greater practical utility and illustrate this with examples from de novo structure prediction, NMR structure calculation, and comparative modeling.     

Biochemical studies of myosin

This article describes methods for expressing and obtaining purified smooth muscle myosin subfragments using the baculovirus/insect cell expression system, as well as methods for purifying whole myosin from tissue. Protocols for several gel assays that are routinely used with myosin are given, including gels to monitor light chain phosphorylation state and native gels to determine protein homogeneity. Steady-state myosin ATPase and actin-activated ATPase determinations are described, as are some of the more basic transient-state kinetic parameters that can be measured. The in vitro motility assay, in which the rate of actin movement over myosin or its subfragments is quantified, is also presented.     

X-ray analysis of the single chain B29-A1 peptide-linked insulin molecule. A completely inactive analogue.

A crystal structure of a totally inactive insulin molecule has been determined. For this insulin molecule, the first without detectable activity to be characterized, the A and B-chains are linked by a peptide bond between A1 Gly and B29 Lys. The molecule has retained all its normal self-association properties and it can also accommodate the two different conformations designated T and R, as seen in 4Zn native pig insulin crystals. The hexamers of the crosslinked insulin molecule were crystallized using the 4Zn insulin recipe of Schlichtkrull. The structure has been crystallographically refined with data extending to 2 A using restrained least-square methods. Comparison of the B29-A1 peptide crosslink insulin and the 4Zn native insulin reveals close structural similarities with the native dimer. The analysis of the structure confirms the earlier hypothesis that insulin structures in crystals are not in an active conformation and that a separation of N-terminal A-chain and C-terminal B-chain is required for interaction with the insulin receptor.     

Antipodean aquaculture agents

Parallel features exist among diseases in Antipodean hosts when compared with those in other tropical and temperate regions. These features can be associated between infections in fish and prawns and between infections in freshwater and saltwater systems. Both fish and prawns are prone to physical stress, to behavioural stress, and to both introduced and native disease agents, agents that are limiting factors in several established aquaculture ventures. Both types of hosts are reared in a variety of facilities, each allowing for a different dynamic predisposition for disease. These diseases include those caused by native freshwater, estuarine and marine agents in both introduced and native hosts, and by introduced agents in those hosts. Some infections reflect unexpected or unidentifiable agents and host responses. Much can be learned in Australia and New Zealand from the infections, successes, failures and innovative methodologies experienced or used in the rest of the world. Potential in the Antipodes for production of unique and abundant fishery products is immense for both local and export markets. Approaches, however, should remain cautious regarding introductions and persistent regarding the investigation of potential diseases, host responses, diagnostic methods and management techniques.     

Absolute quality evaluation of protein model structures using statistical potentials with respect to the native and reference states

In protein structure prediction, it is crucial to evaluate the degree of native-likeness of given model structures. Statistical potentials extracted from protein structure data sets are widely used for such quality assessment problems, but they are only applicable for comparing different models of the same protein. Although various other methods, such as machine learning approaches, were developed to predict the absolute similarity of model structures to the native ones, they required a set of decoy structures in addition to the model structures. In this paper, we tried to reformulate the statistical potentials as absolute quality scores, without using the information from decoy structures. For this purpose, we regarded the native state and the reference state, which are necessary components of statistical potentials, as the good and bad standard states, respectively, and first showed that the statistical potentials can be regarded as the state functions, which relate a model structure to the native and reference states. Then, we proposed a standardized measure of protein structure, called native-likeness, by interpolating the score of a model structure between the native and reference state scores defined for each protein. The native-likeness correlated with the similarity to the native structures and discriminated the native structures from the models, with better accuracy than the raw score. Our results show that statistical potentials can quantify the native-like properties of protein structures, if they fully utilize the statistical information obtained from the data set.     

Comparison of native and non‐native ubiquitin oligomers reveals analogous structures and reactivities

Ubiquitin (Ub) chains regulate a wide range of biological processes, and Ub chain connectivity is a critical determinant of the many regulatory roles that this post‐translational modification plays in cells. To understand how distinct Ub chains orchestrate different biochemical events, we and other investigators have developed enzymatic and non‐enzymatic methods to synthesize Ub chains of well‐defined length and connectivity. A number of chemical approaches have been used to generate Ub oligomers connected by non‐native linkages; however, few studies have examined the extent to which non‐native linkages recapitulate the structural and functional properties associated with native isopeptide bonds. Here, we compare the structure and function of Ub dimers bearing native and non‐native linkages. Using small‐angle X‐ray scattering (SAXS) analysis, we show that scattering profiles for the two types of dimers are similar. Moreover, using an experimental structural library and atomistic simulations to fit the experimental SAXS profiles, we find that the two types of Ub dimers can be matched to analogous structures. An important application of non‐native Ub oligomers is to probe the activity and selectivity of deubiquitinases. Through steady‐state kinetic analyses, we demonstrate that different families of deubiquitinases hydrolyze native and non‐native isopeptide linkages with comparable efficiency and selectivity. Considering the significant challenges associated with building topologically diverse native Ub chains, our results illustrate that chains harboring non‐native linkages can serve as surrogate substrates for explorations of Ub function.     

Catalysis of proline isomerization during protein-folding reactions

The enzyme peptidylprolyl cis-trans isomerase (PPI) is known to catalyze proline isomerization in short proline-containing peptides. If PPI can be shown to generally catalyze isomerization of proline residues in proteins, then it would be a valuable diagnostic reagent for recognition of isomerization, which has proven to be extremely difficult to characterize by other methods. In this study, the catalytic effect of PPI on the slow refolding reactions of seven different proteins has been studied, and in only two cases (RNase T1 and cytochrome c) could significant catalysis be seen. PPI also caused no enhancement in the rate for the 'subtle' conformational changes of native concanavalin A or native Fragment I of prothrombin, which have been suggested to be rate-limited by proline isomerization. There was a small effect of PPI observed for the generation of native RNAase A from the fully-reduced form when the glutathione concentration was low. The conclusion from these studies is that PPI can weakly catalyze some protein processes which are rate-limited by proline isomerization, but probably exhibits no measureable catalysis toward others. This somewhat limits the usefulness of PPI as a diagnostic reagent for proline isomerization.