Solution structure, divalent metal and DNA binding of the endonuclease domain from the replication initiation protein from porcine circovirus 2

Circoviruses are the smallest circular single-stranded DNA viruses able to replicate in mammalian cells. Essential to their replication is the replication initiator, or Rep protein that initiates the rolling circle replication (RCR) of the viral genome. Here we report the NMR solution three-dimensional structure of the endonuclease domain from the Rep protein of porcine circovirus type 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome in swine. The domain comprises residues 12-112 of the full-length protein and exhibits the fold described previously for the Rep protein of the representative geminivirus tomato yellow leaf curl Sardinia virus. The structure, however, differs significantly in some secondary structure elements that decorate the central five-stranded beta-sheet, including the replacement of a beta-hairpin by an alpha-helix in PCV2 Rep. The identification of the divalent metal binding site was accomplished by following the paramagnetic broadening of NMR amide signals upon Mn(2+) titration. The site comprises three conserved acidic residues on the exposed face of the central beta-sheet. For the 1:1 complex of the PCV2 Rep nuclease domain with a 22mer double-stranded DNA oligonucleotide chemical shift mapping allowed the identification of the DNA binding site on the protein and aided in constructing a model of the protein/DNA complex.     

A comparison of the folding of two knotted proteins: YbeA and YibK

The extraordinary topology of proteins belonging to the alpha/beta-knot superfamily of proteins is unexpected, due to the apparent complexities involved in the formation of a deep trefoil knot in a polypeptide backbone. Despite this, an increasing number of knotted structures are being identified; how such proteins fold remains a mystery. Studies on the dimeric protein YibK from Haemophilus influenzae have led to the characterisation of its folding pathway in some detail. To complement research into the folding of YibK, and to address whether folding pathways are conserved for members of the alpha/beta-knot superfamily, the structurally similar knotted protein YbeA from Escherichia coli has been studied. A comprehensive thermodynamic and kinetic analysis of the folding of YbeA is presented here, and compared to that of YibK. Both fold via an intermediate state populated under equilibrium conditions that is monomeric and considerably structured. The unfolding/refolding kinetics of YbeA are simpler than those found for YibK and involve two phases attributed to the formation of a monomeric intermediate state and a dimerisation step. In contrast to YibK, a change in the rate-determining step on the unfolding pathway for YbeA is observed with a changing concentration of urea. Despite this difference, both proteins fold by a mechanism involving at least one sequential monomeric intermediate that has properties similar to that observed during the equilibrium unfolding. The rate of dimerisation observed for YbeA and YibK is very similar, as is the rate constant for formation of the kinetic monomeric intermediate that precedes dimerisation. The findings suggest that relatively slow folding and dimerisation may be common attributes of knotted proteins.     

Shape variation in protein binding pockets and their ligands

A common assumption about the shape of protein binding pockets is that they are related to the shape of the small ligand molecules that can bind there. But to what extent is that assumption true? Here we use a recently developed shape matching method to compare the shapes of protein binding pockets to the shapes of their ligands. We find that pockets binding the same ligand show greater variation in their shapes than can be accounted for by the conformational variability of the ligand. This suggests that geometrical complementarity in general is not sufficient to drive molecular recognition. Nevertheless, we show when considering only shape and size that a significant proportion of the recognition power of a binding pocket for its ligand resides in its shape. Additionally, we observe a "buffer zone" or a region of free space between the ligand and protein, which results in binding pockets being on average three times larger than the ligand that they bind.     

Crystal structures of the luciferase and green fluorescent protein from Renilla reniformis

Due to its ability to emit light, the luciferase from Renilla reniformis (RLuc) is widely employed in molecular biology as a reporter gene in cell culture experiments and small animal imaging. To accomplish this bioluminescence, the 37-kDa enzyme catalyzes the degradation of its substrate coelenterazine in the presence of molecular oxygen, resulting in the product coelenteramide, carbon dioxide, and the desired photon of light. We successfully crystallized a stabilized variant of this important protein (RLuc8) and herein present the first structures for any coelenterazine-using luciferase. These structures are based on high-resolution data measured to 1.4 Å and demonstrate a classic α/β-hydrolase fold. We also present data of a coelenteramide-bound luciferase and reason that this structure represents a secondary conformational form following shift of the product out of the primary active site. During the course of this work, the structure of the luciferase's accessory green fluorescent protein (RrGFP) was also determined and shown to be highly similar to that of Aequorea victoria GFP.     

Quantitative analysis of glutamatergic and GABAergic neurons expressing 5-HT(2A) receptors in human and monkey prefrontal cortex

5-hydroxytryptamine (5-HT) or serotonin 2A receptors play an important role in modulation of prefrontal cortex (PFC) activity and have been implicated in the physiopathology of psychiatric disorders. There is no quantitative information on the percentage of glutamatergic and GABAergic cells that express 5-HT(2A) receptors in human and monkey PFC. We have used double in situ hybridization to quantify the mRNA co-localization of 5-HT(2A) receptor with the glutamatergic transporter vesicular glutamate transporter 1, and with the GABAergic marker glutamic acid decarboxylase 65/67 and in parvalbumin and calbindin GABAergic cell populations. Our results show that nearly every glutamatergic cell (86-100%) in layers II-V expressed 5-HT(2A) receptor mRNA in both species. This percentage was lower in layer VI (13-31%). In contrast, not all the GABAergic interneurons (13-46%) expressed 5-HT(2A) receptor mRNA. This receptor was expressed in 45-69% of parvalbumin and in 61-87% of calbindin positive cells. These results indicate that, while the majority of glutamatergic neurons can be sensitive to 5-HT action via 5-HT(2A) receptors, this modulation occurs only in a limited population of GABAergic interneurons and provides new neuroanatomical information about the role played by serotonin through 5-HT(2A) receptors in the PFC and on the sites of action for drugs such as antipsychotics and antidepressants used in treatment of psychiatric disorders.     

Landscape Analysis of Bobcat Habitat in the Northern Lower Peninsula of Michigan

ABSTRACT Controversy over bobcat (Lynx rufus) management in the northern Lower Peninsula of Michigan (NLP), USA, stimulated a need for information on the distribution of Michigan bobcats. From March 2003 to October 2004, we conducted a radiotelemetry and scentstation survey study of bobcats in the NLP. We developed a spatial model to predict bobcat distribution throughout the NLP based on bobcat area requirements, habitat and landscape variables derived from remotely sensed land-cover data, and a multivariate distance statistic. Bobcat 50% minimum convex polygon core areas were comprised of more lowland forest (51%), nonforested wetlands (9%), and streams (3%) than the surrounding NLP. The NLP was comprised primarily of upland forest (44%) and field (32%). Habitat in the northeast and central regions of the NLP was most similar to the habitat composition of bobcat core areas. This model will be useful in aiding Michigan wildlife management agencies with assessing the status and distribution of the NLP bobcat population by identifying areas important to bobcats and supporting the development of regional strategies for carnivore conservation.     

蚯蚓对不同阔叶落叶的摄食作用

采取室内试验,研究蚯蚓对红松阔叶混交林中几种不同阔叶的摄食量及影响因素、蚯蚓在不同阔叶中的生长状况和对有机碳和全氮的影响,探讨蚯蚓在东北红松阔叶混交林阔叶分解中的作用。蚯蚓喜食枫桦、紫椴和色木槭分解落叶,不喜食水曲柳分解落叶。试验表明,温度、凋落叶种类和凋落叶的分解时间等因素影响蚯蚓摄食量的大小。蚯蚓对3种喜食分解落叶摄食量平均值为:枫桦>色木槭>紫椴。不同温度条件下蚯蚓摄食量平均值为:20℃>25℃>15℃。凋落叶分解时间越长,蚯蚓越喜食,其摄食量也越大。分解落叶的C/N比新鲜凋落叶低。蚯蚓在3种凋落叶中的日增重倍数和日排粪量随着温度的升高而增加。不同温度下蚯蚓日增重倍数的平均值为25℃>20℃>15℃。蚯蚓在紫椴凋落叶中日增重倍数高于枫桦和色木槭分解落叶,在色木槭分解落叶的摄食过程中日排粪平均值高于紫椴和枫桦凋落叶。微生物对落叶中有机碳分解40d的作用小于蚯蚓对落叶摄食20d。蚯蚓摄食20d对增加凋落叶中的全氮含量的作用大于微生物单独分解40d。在凉水国家级自然保护区,蚯蚓对阔叶凋落叶的分解占年平均凋落叶量的10.3%左右。蚯蚓在红松阔叶混交林中阔叶分解中的作用不可忽视。