Protein folding pathways of adenylate kinase from E. coli: hydrostatic pressure and stopped-flow studies.

Adenylate kinase (AKe) from E. coli is a small, single-chain, monomeric enzyme with no tryptophan and a single cysteine residue. We have constructed six single-Trp mutants of AKe to facilitate optical studies of these proteins and to specifically examine the interrelationship between their structure, function, dynamics, and folding reactions. In this study, the effects of hydrostatic pressure on the folding reactions of AKe were studied. The native structure of AKe was transformed to a non-native, yet pressure stable, conformation by hydrostatic pressure of about 300 MPa. This pressure lability of AKe is rather low for a monomeric protein and presumably may be attributed to substantial conformational flexibility and a correspondingly large volume change. The refolding of AKe after pressure-induced denaturation was reversible under ambient conditions. At low temperature (near 0 degrees C), the refolding process of pressure-exposed AKe mutants displayed a significant hysteresis. The observation of a slow refolding rate in the 193 region and a faster folding rate around the active site (86, 41, 73 regions) leads us to suggest that in the folding process, priority is afforded to functional regions. The slow structural return of the 193 region apparently does not hinder the more rapid return of enzymatic activity of AKe. Circular dichroism studies on the pressure-denatured Y193W mutant show that the secondary structure (calculated from far-UV spectra) returned at a rapid rate, but the tertiary structure alignment (calculated from near-UV spectra) around the 193 region occurred more slowly at rates comparable to those detected by fluorescence intensity. Denaturation of AKe mutants by guanidine hydrochloride and subsequent refolding experiments were also consistent with a much slower refolding process around the 193 region than near the active site. Fast refolding kinetic traces were observed in F86W, S41W, and A73W mutants using a fluorescence detection stopped-flow rapid mixing device, while only a slow kinetic trace was observed for Y193W. The results suggest that the differences in regional folding rates of AKe are not derived from the specific denaturation methods, but rather are inherent in the structural organization of the protein.     

Codon usage in the vertebrate hemoglobins and its implications

A study of codon usage in vertebrate hemoglobins revealed an evolutionary trend toward elevated numbers of CpG codon boundary pairs in mammalian hemoglobin alpha genes. Selection for CpG codon boundaries countering the generally observed CpG suppression is strongly suggested by these data. These observations parallel recently published experimental results that indicate that constitutive expression of the human alpha-globin gene appears to be determined by regulatory information encoded within the structural gene. The possibility is raised that, in the absence of selection, CpG decay can be used to date the evolutionary origin of a mammalian alpha pseudogene from its active alpha gene.      

Evolution of crystallins: expression of lens-specific proteins in the blind mammals mole (Talpa europaea) and mole rat (Spalax ehrenbergi)

The mole (Talpa europaea; Insectivora) and the mole rat (Spalax ehrenbergi; Rodentia) both have degenerated eyes as a convergent adaptation to subterranean life. The rudimentary eye lenses of these blind mammals no longer function in a visual process. The crystallin genes, which display a lens-specific expression pattern, were studied in these blind mammals and in related species with normal eyes by hybridizing their genomic DNAs with probes obtained from cDNA clones for alpha A-, alpha B-, and beta Bp-crystallins from calf and gamma 3- crystallin from the rat. For all crystallin genes examined, the hybridization signals of mole and mole rat genomic DNA were comparable, respectively, with those of shrew and of rat and mouse, normal-vision representatives of the orders Insectivora and Rodentia. The expression of the crystallins at the protein level was tested by using antiserum specific for alpha-crystallin in immunofluorescence reactions on lens sections of mole and mole rat eyes and by using antisera against the beta- and gamma-crystallins on sections of the mole eye. All antisera gave positive fluorescence reactions exclusively with lens tissue of these blind mammals, indicating that the crystallins are still normally expressed despite the fact that these lenses have had no function in a visual process in these mammals for at least many million years. These findings apparently imply that some unknown selective advantage has conserved the crystallin genes and their expression after the loss of normal function of the lenses.      

The evolutionary history of the amylase multigene family in Drosophila pseudoobscura

In Drosophila pseudoobscura, the amylase (Amy) multigene family is contained within a series of inversions, or gene arrangements, on the third chromosome. The Standard (ST), Santa Cruz (SC), and Tree Line (TL) inversions are central to the phylogeny of arrangements, and have clusters of other arrangements derived from them. The gene arrangements belonging to each of these three clusters have a characteristic number of Amy genes, ranging from three in ST to two in SC to one in TL. This distribution pattern can reflect a history of either duplications or deletions, although the data available in the past did not permit a decision between these alternatives. We provide unambiguous evidence that three Amy genes were present before the divergence of the ST, SC, and TL arrangements. Thus, the current status of the Amy multigene family is the result of deletions in the TL and SC arrangements, which created three new pseudogenes: TL Amy2-psi, TL Amy3-psi, and SC Amy3- psi. Analysis of pseudogene sequences revealed that, in the SC and ST arrangements, pseudogene evolution has been retarded, most likely due to the homogenization effect of gene conversion. Finally, by determining the original copy number, we have reconstructed the evolutionary history of the Amy multigene family and linked it with the evolution of the central gene arrangements.      

Evidence for gene conversion in the amylase multigene family of Drosophila pseudoobscura

The alpha-amylase (Amy) multigene family in Drosophila pseudoobscura is located on the third chromosome, which is polymorphic for more than 40 inverted gene arrangements. The number of copies in this family ranges from one to three, depending on the arrangement in question. A previous study of the three Amy genes from the Standard (ST) arrangement suggested either that duplicated copies (Amy2 and Amy3) are functionally constrained or that they are undergoing gene conversion with Amy1. In order to elucidate further the pattern of molecular evolution in this family, we cloned and sequenced four additional Amy genes, two from the Santa Cruz (SC) and two from the Chiricahua (CH) gene arrangement. Of the two alternatives, only the hypothesis of gene conversion is supported by the sequence analysis. The homogenization effect of gene conversion has been strongest in SC, whose copies differ by only two nucleotides, less noticeable in ST, and negligible in the CH. Furthermore, the action of gene conversion is apparently localized, occurring only in the coding region. Interestingly, these results concur with the findings of other workers for the duplicated Amy genes in the Drosophila melanogaster group. Thus, the occurrence of gene conversion in the Amy multigene family seems to be a common feature in the Drosophila species studied so far.      

Expression,secretion and surface display of a human alkaline phosphatase by the ciliate <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Background  

Tetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites Ichthyophthirius multifiliis and Plasmodium falciparum and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that T. thermophila is also able to produce a functional human DNase I. The present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase (hiAP) using T. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system.     

Spatial-temporal studies of membrane dynamics: scanning fluorescence correlation spectroscopy (SFCS)

Giant unilamellar vesicles (GUVs) have been widely used as a model membrane system to study membrane organization, dynamics, and protein-membrane interactions. Most recent studies have relied on imaging methods, which require good contrast for image resolution. Multiple sequential image processing only detects slow components of membrane dynamics. We have developed a new fluorescence correlation spectroscopy (FCS) technique, termed scanning FCS (i.e., SFCS), which performs multiple FCS measurements simultaneously by rapidly directing the excitation laser beam in a uniform (circular) scan across the bilayer of the GUVs in a repetitive fashion. The scan rate is fast compared to the diffusion of the membrane proteins and even small molecules in the GUVs. Scanning FCS outputs a "carpet" of timed fluorescence intensity fluctuations at specific points along the scan. In this study, GUVs were assembled from rat kidney brush border membranes, which included the integral membrane proteins. Scanning FCS measurements on GUVs allowed for a straightforward detection of spatial-temporal interactions between the protein and the membrane based on the diffusion rate of the protein. To test for protein incorporation into the bilayers of the GUVs, antibodies against one specific membrane protein (NaPi II cotransporter) were labeled with ALEXA-488. Fluorescence images of the GUVs in the presence of the labeled antibody showed marginal fluorescence enhancement on the GUV membrane bilayers (poor image contrast and resolution). With the application of scanning FCS, the binding of the antibody to the GUVs was detected directly from the analysis of diffusion rates of the fluorescent antibody. The diffusion coefficient of the antibody bound to NaPi II in the GUVs was approximately 200-fold smaller than that in solution. Scanning FCS provided a simple, quantitative, yet highly sensitive method to study protein-membrane interactions.     

Multimodal CARS microscopy determination of the impact of diet on macrophage infiltration and lipid accumulation on plaque formation in ApoE-deficient mice

We characterized several cellular and structural features of early stage Type II/III atherosclerotic plaques in an established model of atherosclerosis—the ApoE-deficient mouse—by using a multimodal, coregistered imaging system that integrates three nonlinear optical microscopy (NLOM) contrast mechanisms: coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), and two-photon excitation fluorescence (TPEF). Specifically, the infiltration of lipid-rich macrophages and the structural organization of collagen and elastin fibers were visualized by CARS, SHG, and TPEF, respectively, in thick tissue specimens without the use of exogenous labels or dyes. Label-free CARS imaging of macrophage accumulation was confirmed by histopathology using CD68 staining. A high-fat, high-cholesterol Western diet resulted in an approximate 2-fold increase in intimal plaque area, defined by CARS signals of lipid-rich macrophages. Additionally, analysis of collagen distribution within lipid-rich plaque regions revealed nearly a 4-fold decrease in the Western diet–fed mice, suggesting NLOM sensitivity to increased matrix metalloproteinase (MMP) activity and decreased smooth muscle cell (SMC) accumulation. These imaging results provide significant insight into the structure and composition of early stage Type II/III plaque during formation and allow for quantitative measurements of the impact of diet and other factors on critical plaque and arterial wall features.     

Fluorescence quenching studies of apolipoprotein A-I in solution and in lipid-protein complexes: protein dynamics

Fluorescence lifetime and intensity quenching studies of human plasma apolipoprotein A-I (apo A-I) in aqueous solution and in recombinant lipoprotein complexes with dimyristoylphosphatidylcholine (DMPC) indicate differences in conformational dynamics. In aqueous solution, the bimolecular quenching constants (k*) for lipid-free apo A-I fluorescence quenching by oxygen and acrylamide are 2.4 X 10(9) and 0.38 X 10(9) M-1 s-1, respectively. These values are independent of the oligomeric form of the protein. There is no correlation between the relatively small k* for apo A-I, which reflects rapid, low-amplitude protein fluctuations, and the labile conformational changes of apo A-I folding reactions, like denaturation, which occur on a slower time scale. In recombinant DMPC/apo A-I complexes (100:1 molar ratio) the protein increases in amphiphilic alpha-helical structure as it blankets the lipid matrix. The apparent k* for oxygen quenching of apo A-I fluorescence in the complex is large and increases in a temperature-dependent manner. We have introduced a two-compartment model, which discriminates the source of quencher molecules as aqueous or lipid, to describe oxygen quenching of DMPC/apo A-I fluorescence. The magnitude and temperature dependence of the apparent k* predominantly reflect the partitioning of oxygen between the two phases rather than being a probe of the lipid physical state. Calculations of the helical hydrophobic moment in apo A-I indicate that tryptophan residues 8 and 72 occur at the lipid-protein interface of amphiphilic alpha-helices, whereas the other two tryptophan residues (50, 108) lie on the nonpolar faces of amphiphilic helices.(ABSTRACT TRUNCATED AT 250 WORDS)     

代谢、血液碱化和纯氧影响呼吸调控的人体实验研究II： 血液碱化后运动试验*

目的: 在完成吸入室内空气状态下症状限制性最大极限心肺运动试验(CPET)和动脉血气指标动态变化规律的基础上,进一步探讨体液酸碱度和CO₂含量对呼吸调控的影响。方法: 选正常志愿者5名,给予5%NaHCO₃(总量0.3 g/kg)分次口服,每5 min口服75 ml(3.75g )。总量服完1 h后,重复CPET。于静息、热身、运动及恢复期,连续测定肺通气指标及每分钟动脉取样的血气指标变化,并与本人在非碱化血液条件下对照数据进行配对t检验比较。结果: 碱化血液之后,CPET期间随着运动功率逐步递增,气体交换和血气指标的反应模式与非碱化血液对照相似（P>0.05）;即与静息状态比较,每分通气量、潮气量、呼吸频率、VO₂、VCO₂均呈现近于线性渐进性递增(P<0.05~0.001)。与碱化血液前吸入室内空气的对照比较：在碱化血液条件下,所有时间点血红蛋白浓度,PaCO₂与pH均显著提高(P<0.05);除无氧阈PaCO₂减低外,只有热身状态呈增高态势,统计学有显著差异(P<0.05);而PaO₂无差异(P>0.05),各状态均较对照状态减低,除恢复期外均有统计学差异(P<0.05)。与非碱化血液对照比较,除静息每分通气量低于对照(P<0.05）外,所有通气指标均无统计学差异(P>0.05)。结论: 碱化血液条件下, 尽管有更高的CaCO₂, PaCO₂ 和 pHa平均水平及更低的Hba和[H⁺]a平均水平,机体对CPET的呼吸反应模式基本相似。