Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling.

An unresolved key issue in the mechanism of protein folding assisted by the molecular chaperone GroEL is the nature of the substrate protein bound to the chaperonin at different stages of its reaction cycle. Here we describe the conformational properties of human dihydrofolate reductase (DHFR) bound to GroEL at different stages of its ATP-driven folding reaction, determined by hydrogen exchange labeling and electrospray ionization mass spectrometry. Considerable protection involving about 20 hydrogens is observed in DHFR bound to GroEL in the absence of ATP. Analysis of the line width of peaks in the mass spectra, together with fluorescence quenching and ANS binding studies, suggest that the bound DHFR is partially folded, but contains stable structure in a small region of the polypeptide chain. DHFR rebound to GroEL 3 min after initiating its folding by the addition of MgATP was also examined by hydrogen exchange, fluorescence quenching, and ANS binding. The results indicate that the extent of protection of the substrate protein rebound to GroEL is indistinguishable from that of the initial bound state. Despite this, small differences in the quenching coefficient and ANS binding properties are observed in the rebound state. On the basis of these results, we suggest that GroEL-assisted folding of DHFR occurs by minor structural adjustments to the partially folded substrate protein during iterative cycling, rather than by complete unfolding of this protein substrate on the chaperonin surface.     

Ligand recognition in μ opioid receptor: experimentally based modeling of μ opioid receptor binding sites and their testing by ligand docking

For three-dimensional understanding of the mechanisms that control potency and selectivity of the ligand binding at the atomic level, we have analysed opioid receptor-ligand interaction based on the receptor's 3D model. As a first step, we have constructed molecular models for the multiple opioid receptor subtypes using bacteriorhodopsin as a template. The S-activated dihydromorphine derivatives should serve as powerful tools in mapping the three-dimensional structure of the μ opioid receptor, including the nature of the agonist-mediated conformational change that permits G protein-coupling to ‘second messenger’ effector molecules, and in identifying specific ligand-binding contacts with the μ opioid receptor. The analyses of the interactions of some opioid ligands with the predicted ligand binding sites are consistent with the results of the affinity labeling experiments.     

DNA水平上的植物系统学研究方法

本文简要总结了近年来在ＤＮＡ水平上的植物系统学研究方法,着重介绍了限制性长度多态性分析,ＰＣＲ技术在植物系统学上的应用等这一领域最新的进展,并对分子数据的分析方法及系统树的构建进行了详细讨论。     

鼠抗人纤维蛋白抗体单链Fv片断的三维结构模建

用Biosym公司开发的计算机辅助分子设计系统模建了鼠抗人纤维蛋白抗体Fv片断的三维结构。Fv是由重链可变区(Vh)和轻链可变区(Vl)两个结构域组成的具有抗原结合能力的最小抗体片断。先分别模建了Vh和Vl两个结构域,然后搭建出Fv片断的整体三维结构,并对模建的结构进行了分子力学和动力学优化。对结构的合理性验证显示模建结构是合理的。本研究为鼠抗人纤维蛋白抗体Fv片断的人源化的分子设计打下了基础。     

Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies

Eukaryotic proteins expressed inEscherichia coli often accumulate within the cell as insoluble protein aggregates or inclusion bodies. The recovery of structure and activity from inclusion bodies is a complex process, there are no general rules for efficient renaturation. Research into understanding how proteins fold in vivo is giving rise to potentially new refolding methods, for example, using molecular chaperones. In this article we review what is understood about the main three classes of chaperone: the Stress 60, Stress 70, and Stress 90 proteins. We also give an overview of current process strategies for renaturing inclusion bodies, and report the use of novel developments that have enhanced refolding yields.     

Phylogenetic analysis of cytochromeb sequences in the dasyurid marsupial subfamily phascogalinae: Systematics and the evolution of reproductive strategies

We report DNA sequence variation in 861 bp of the mitochondrial cytochromeb gene from 10 species of the dasyurid marsupial subfamily Phascogalinae (including the New Guinean genusMurexia) and an outgroup planigale (Planigale ingrami). Phylogenetic analyses of these sequences indicate that (1) the subfamily consists of three major clades corresponding to (a)Phascogale, (b) AustralianAntechinus, and (c) New Guinean Antechinus andMurexia; (2) Antechinus habbema constitutes the earliest branch of the New Guinean clade; and (3); Antechinus melanurus and A. naso are sister species within the New Guinean clade. Among Australian antechnuses,A. stuartii andA. swainsonii are more closely related to each other than either is toA. flavipes, a result that is seemingly at odds with all previous systematic studies. Although resolution is limited, it appears thatAntechnius andMurexia species form a clade to the exclusion ofPhascogale. This relationship suggests that male semelparity is not a strong synapomorphy for Australian antechinuses and phascogales, despite its apparent physiological similarity in the two groups.To whom correspondence should be addressed.     

Nitric oxide is involved in heat-induced HSP70 accumulation

Heat shock potentiated the nitric oxide production (EPR assay) in the liver, kidney, heart, spleen, intestine, and brain. The heat shock-induced sharp transient increase in the rate of nitric oxide production preceded the accumulation of heat shock proteins (HSP70) (Western blot analysis) as measured in the heart and liver. In all organs the nitric oxide formation was completely blocked by the NO-synthase inhibitor (L-NNA). L-NNA also markedly attenuated the heat shock-induced accumulation of HSP70. The results suggests that nitric oxide is involved in the heat shock-induced activation of HSP70 synthesis.     

Male mating success, reproductive success and multiple paternity in a natural population of sand lizards: behavioural and molecular genetics data

Sand lizard Lacerta agilis females characteristically mate with several males which, in staged mating experiments, results in multiple paternity of the offspring. In order to investigate multiple paternity in a natural population and interpret male reproductive behaviours in terms of sired young, we sampled the blood of females, potential fathers and hatchlings, and determined paternity using multilocus DNA fingerprinting as well as the variation at a single locus detected by the probe (TC) _n . The paternity analyses were preceded by a laboratory experiment in which we established that the parental alleles identified by the single locus probe were inherited in a Mendelian way. Our molecular data demonstrated that 12 out of 13 males (92%) that sired offspring were correctly identified from the 56 sexually mature males in the population. Also smaller males were accepted as sexual partners by the females, but sired fewer young in competition with larger males and were less able to maintain prolonged post-copulatory mate guarding. This may result in that some sexually successful males are only observed inside a female's home range, but never in pair-association with the female.     

Crystal structures of bovine chymotrypsin and trypsin complexed to the inhibitor domain of Alzheimer's amyloid beta-protein precursor (APPI) and basic pancreatic trypsin inhibitor (BPTI): engineering of inhibitors with altered specificities.

The crystal structures of the inhibitor domain of Alzheimer's amyloid beta-protein precursor (APPI) complexed to bovine chymotrypsin (C-APPI) and trypsin (T-APPI) and basic pancreatic trypsin inhibitor (BPTI) bound to chymotrypsin (C-BPTI) have been solved and analyzed at 2.1 A, 1.8 A, and 2.6 A resolution, respectively. APPI and BPTI belong to the Kunitz family of inhibitors, which is characterized by a distinctive tertiary fold with three conserved disulfide bonds. At the specificity-determining site of these inhibitors (P1), residue 15(I)4 is an arginine in APPI and a lysine in BPTI, residue types that are counter to the chymotryptic hydrophobic specificity. In the chymotrypsin complexes, the Arg and Lys P1 side chains of the inhibitors adopt conformations that bend away from the bottom of the binding pocket to interact productively with elements of the binding pocket other than those observed for specificity-matched P1 side chains. The stereochemistry of the nucleophilic hydroxyl of Ser 195 in chymotrypsin relative to the scissile P1 bond of the inhibitors is identical to that observed for these groups in the trypsin-APPI complex, where Arg 15(I) is an optimal side chain for tryptic specificity. To further evaluate the diversity of sequences that can be accommodated by one of these inhibitors, APPI, we used phage display to randomly mutate residues 11, 13, 15, 17, and 19, which are major binding determinants. Inhibitors variants were selected that bound to either trypsin or chymotrypsin. As expected, trypsin specificity was principally directed by having a basic side chain at P1 (position 15); however, the P1 residues that were selected for chymotrypsin binding were His and Asn, rather than the expected large hydrophobic types. This can be rationalized by modeling these hydrophilic side chains to have similar H-bonding interactions to those observed in the structures of the described complexes. The specificity, or lack thereof, for the other individual subsites is discussed in the context of the "allowed" residues determined from a phage display mutagenesis selection experiment.