Disulfide trapping of protein complexes on the yeast surface

Protein complexes are common in nature and play important roles in biology, but studying the quaternary structure formation in vitro is challenging since it involves lengthy and expensive biochemical steps. There are frequent technical difficulties as well with the sensitivity and resolution of the assays. In this regard, a technique that can analyze protein–protein interactions in high throughput would be a useful experimental tool. Here, we introduce a combination of yeast display and disulfide trapping that we refer to as stabilization of transient and unstable complexes by engineered disulfide (STUCKED) that can be used to detect the formation of a broad spectrum of protein complexes on the yeast surface using fluorescence labeling. The technique uses an engineered intersubunit disulfide to covalently crosslink the subunits of a complex, so that the disulfide‐trapped complex can be displayed on the yeast surface for detection and analysis. Transient protein complexes are difficult to display on the yeast surface, since they may dissociate before they can be detected due to a long induction period in yeast. To this end, we show that three different quaternary structures with the subunit dissociation constant K_d ～ 0.5–20 µM, the antibody variable domain (Fv), the IL‐8 dimer, and the p53–MDM2 complex, cannot be displayed on the yeast surface as a noncovalent complex. However, when we introduce an interchain disulfide between the subunits, all three systems are efficiently displayed on the yeast surface, showing that disulfide trapping can help display protein complexes that cannot be displayed otherwise. We also demonstrate that a disulfide forms only between the subunits that interact specifically, the displayed complexes exhibit functional characteristics that are expected of wt proteins, the mutations that decrease the affinity of subunit interaction also reduce the display efficiency, and most of the disulfide stabilized complexes are formed within the secretory pathway during export to the surface. Disulfide crosslinking is therefore a convenient way to study weak protein association in the context of yeast display. Biotechnol. Bioeng. 2010; 106: 27–41. © 2009 Wiley Periodicals, Inc.     

PALSSE: A program to delineate linear secondary structural elements from protein structures

Background  

The majority of residues in protein structures are involved in the formation of α-helices and β-strands. These distinctive secondary structure patterns can be used to represent a protein for visual inspection and in vector-based protein structure comparison. Success of such structural comparison methods depends crucially on the accurate identification and delineation of secondary structure elements.     

Evidence of Sundaland’s subsidence requires revisiting its biogeography

It is widely accepted that sea level changes intermittently inundated the Sunda Shelf throughout the Pleistocene, separating Java, Sumatra and Borneo from the Malay Peninsula and from each other. On this basis, the dynamics of the biodiversity hotspot of Sundaland is consistently regarded as solely contingent on glacial sea level oscillations, with interglacial highstands creating intermittent dispersal barriers between disjunct landmasses. However, recent findings on the geomorphology of the currently submerged Sunda shelf suggest that it subsided during the Pleistocene and that, over the Late Pliocene and Quaternary, is was never submerged prior to Marine Isotope Stage 11 (MIS 11, 400 ka). This would have enabled the dispersal of terrestrial organisms regardless of sea level variations until 400 ka and hampered movements thereafter, at least during interglacial periods. Existing phylogeographic data for terrestrial organisms conform to this scenario: available divergence time estimates reveal an 8- to 9-fold increase in the rate of vicariance between landmasses of Sundaland after 400 ka, corresponding to the onset of episodic flooding of the Sunda shelf. These results highlight how reconsidering the paleogeographic setting of Sundaland challenges understanding the mechanisms generating Southeast Asian biodiversity.     

Expression of Tropomyosin Genes During the Development of the Rat Cerebellum

The expression of the tropomyosin genes in the rat nervous system was examined during the postnatal development of the cerebellum, using human-specific alpha-, beta-, gamma-, and delta-tropomyosin cDNA probes and rat-specific alpha-, beta-, and delta-tropomyosin oligonucleotide probes. The beta- and gamma-genes do not seem to be expressed in the rat brain. The delta-tropomyosin gene produces two mRNAs: a major one of 2.4 kb, which is highly concentrated during the first postnatal week and then decreases fourfold in level until the age of 35 days, and a minor one of 2 kb, with the same developmental profile as the 2.4-kb mRNA. A 3-kb mRNA is expressed by the alpha-tropomyosin gene and is characteristic of the mature rat. The expression of the tropomyosin genes during the development of the rat cerebellum does not seem to be regulated through alternative splicing but rather implies the differential expression of two different isogenes. The multiple isoforms of tropomyosin produced during neuronal differentiation may be intimately involved in the regulation of the organization and function of actin microfilaments.     

Structural drift: a possible path to protein fold change

SUMMARY: Along with their mutating sequences, protein structures change in time. Analyzing a formate dehydrogenase domain that is evolutionarily related to ferredoxin, but simultaneously contains all the structural elements of a beta-Grasp fold, we illustrate here a mechanism termed as structural drift, by which changes to a protein fold can occur. CONTACT: grishin@chop.swmed.edu.     

The Epidemiology of Soil-Transmitted Helminths in Bihar State,India

Background

Soil-transmitted helminths (STHs) infect over a billion individuals worldwide. In India, 241 million children are estimated to need deworming to avert the negative consequences STH infections can have on child health and development. In February-April 2011, 17 million children in Bihar State were dewormed during a government-led school-based deworming campaign. Prior to programme implementation, a study was conducted to assess STH prevalence in the school-age population to direct the programme. The study also investigated risk factors for STH infections, including caste, literacy, and defecation and hygiene practices, in order to inform the development of complementary interventions.

Methods

A cross-sectional survey was conducted among children in 20 schools in Bihar. In addition to providing stool samples for identification of STH infections, children completed a short questionnaire detailing their usual defecation and hand-hygiene practices. Risk factors for STH infections were explored.

Results

In January-February 2011, 1279 school children aged four to seventeen provided stool samples and 1157 children also completed the questionnaire. Overall, 68% of children (10-86% across schools) were infected with one or more soil-transmitted helminth species. The prevalence of ascariasis, hookworm and trichuriasis was 52%, 42% and 5% respectively. The majority of children (95%) practiced open defecation and reported most frequently cleansing hands with soil (61%). Increasing age, lack of maternal literacy and certain castes were independently associated with hookworm infection. Absence of a hand-washing station at the schools was also independently associated with A. lumbricoides infection.

Conclusions

STH prevalence in Bihar is high, and justifies mass deworming in school-aged children. Open defecation is common-place and hands are often cleansed using soil. The findings reported here can be used to help direct messaging appropriate to mothers with low levels of literacy and emphasise the importance of water and sanitation in the control of helminths and other diseases.     

The development of wild immature Sumatran orangutans (Pongo abelii) at Ketambe

Orangutans have the longest immature period and inter-birth interval of all ape species. This may be explained by a slow life history, the need to develop skills or by their relatively solitary lifestyle, which prevents a mother from associating with two offspring. This study of wild immature orangutans at the Ketambe Research Station, Indonesia, describes, with partly cross-sectional, partly longitudinal data, their development to independence. The study subjects ranged from 1 to 11 years of age. Data on their activity budget, diet, mother–offspring proximity and maintenance of proximity, association with conspecifics and play behavior were collected. The results indicate that immature orangutans can provide for their own food and transport, and therefore were independent of direct maternal care, at an age of possibly 3 but more clearly 6 years. This is similar to chimpanzees, and refutes the slow life history hypothesis. Immature orangutans remain within their mother’s vicinity until the age of 8 years, indicating a dependence on indirect maternal care, and this coincides with the period during which the mother does not produce another offspring. A female orangutan seems unable to associate with an older immature while caring for a new infant. This is consistent with the solitary-lifestyle hypothesis and corroborates the results obtained with the Sumatran orangutan population at Suaq Balimbing. However, why an immature depends indirectly on its mother for such a long period remains unclear. It is possible that it needs to develop ecological or social skills or needs the protection of its mother. Unfortunately, no data were available to distinguish between these possibilities.