Experimental errors and their effect on analyzing circular dichroism spectra of proteins

Seven types of error that may interfere with the analysis of protein circular dichroism (CD) spectra for secondary structure are examined. Three of these errors are operational encompassing wavelength synchronization, and proper choice of spectral bandwidth and scan speed. Three are experimental involving intensity adjustments and two sources of baseline shift. The skew baseline shift is analogous to error in CD intensity at short wavelengths due to high sample absorption and low source intensity. The final source of error deals with constrained analyses. We have investigated these types of error to determine how they may be affecting our analysis of protein CD spectra and the role they may play in causing our analyses to fail for some proteins. We find that small errors in the baseline (which are independent of the protein spectrum) will rationalize our poor analyses. Spectroscopists must adopt new standards of precision if sophisticated analyses are to succeed.     

Single base errors in PCR products from avian museum specimens and their effect on estimates of historical genetic diversity

Conservation genetic studies often employ DNA extracts from museum specimens for comparisons with extant populations to monitor temporal changes in genetic diversity. Here, we report on artifact base changes in mitochondrial DNA sequences amplified from relatively recent (≤ 35 years) museum specimens of indigobirds (Vidua spp.). Single base errors were confirmed by replicate sequencing and included both double peaks and artifact substitutions at rates of ∼3 × 10⁻⁴ and ∼1 × 10⁻⁴ per base-pair, respectively, resulting in one or more errors or ambiguities in an 1100 base pair sequence in 21% of 219 samples. Most errors involved C→T changes on the L-strand, presumably due to deamination of cytosine in the template. The error rates encountered here bias comparisons of haplotype number between historical and extant populations, such that the ‘loss’ of artifact haplotypes present in a historical sample could be incorrectly attributed to a population decline or bottleneck. Sequencing errors due to miscoding lesions in template DNA have so far been reported only from ancient and formalin-fixed tissue, but they may also affect relatively recent museum samples, as shown here, and perhaps also non-invasive samples that typically yield low-quality DNA.     

Heterogeneity of rat liver mitochondrial fractions and the effect of tri-iodothyronine on their protein turnover

1. Rat liver mitochondria were separated into heavy, light and fluffy fractions by differential centrifugation under standard conditions. 2. All mitochondrial fractions possessed soluble as well as membrane-bound enzymes typical of mitochondria. 3. The heavy fraction represented the stable mitochondrial structures and the fluffy particles appear to be loosely coupled. 4. The light mitochondrial fraction lacked the ability of coupled phosphorylation. 5. A study of mobility and isoelectric pH indicated a similarity in the basic membrane structure of all the mitochondrial fractions. 6. The turnover rates of proteins in the heavy and fluffy particles were almost identical; however, this rate was rapid for the light mitochondrial fraction. 7. On treatment with 3,3',5-tri-iodo-l-thyronine, succinoxidase activity was maximally stimulated much earlier in the light mitochondrial fraction than in the heavy fraction. The activity of the fluffy particles, however, remained almost unaffected. 8. Malate dehydrogenase activity in all the mitochondrial fractions was stimulated only at 40h after tri-iodothyronine treatment. 9. The pattern of incorporation of dl-[1-(14)C]leucine in vivo in the tri-iodothyronine-treated animals indicated a rapid initial incorporation and high synthetic ability of the light mitochondrial fraction. 10. The turnover pattern of proteins of the mitochondrial fractions from animals receiving repeated doses of tri-iodothyronine was remarkably different from the normal pattern and suggested that preformed soluble protein units may be incorporated in the light mitochondrial fraction during maturation to form the stable heavy mitochondria. 11. The amount of light-mitochondrial proteins decreased by 40% on thyroidectomy and increased by 160% on treatment with tri-iodothyronine. 12. The possible significance of these results is discussed in relation to mitochondrial genesis.     

The effect of surgical trauma on muscle protein turnover in rats.

The rate of synthesis and catabolism of sarcoplasmic- and myofibrillar-muscle protein was measured in operated, sham-operated and food-restricted rats by using Na2 14CO3. The food-restricted group underwent sham operations and were limited to the food intake of the operated animals. Protein synthesis and catabolism were increased in the sarcoplasmic-muscle fraction in operated rats compared with that in sham-operated or food-restricted rats. The rate of synthesis of the myofibrillar protein decreased in operated animals, but the rate of catabolism was not altered in the myofibrillar-muscle fraction of the operated animals compared with that in food-restricted and sham-operated animals. In the operated animals, there was a net loss of protein from the muscle. Thus the rats that underwent surgery lost muscle protein, primarily as a result of a decrease in synthesis of myofibrillar protein. The changes in protein turnover in operated animals were not due to decreases in food intake, since protein turnover in sham-operated animals that were restricted to the food intake of the operated rats was not different from that in sham-operated rats fed ad libitum.     

The effect of protein depletion and repletion on muscle-protein turnover in the chick.

Rates of growth and protein turnover in the breast muscle of young chicks were measured in order to assess the roles of protein synthesis and degradation in the regulation of muscle mass. Rates of protein synthesis were measured in vivo by injecting a massive dose of L-[1-14C]valine, and rates of protein degradation were estimated as the difference between the synthesis rate and the growth rate of muscle protein. In chicks fed on a control diet for up to 7 weeks of age, the fractional rate of synthesis decreased from 1 to 2 weeks of age and then changed insignificantly from 2 to 7 weeks of age, whereas DNA activity was constant for 1 to 7 weeks. When 4-week-old chicks were fed on a protein-free diet for 17 days, the total amount of breast-muscle protein synthesized and degraded per day and the amount of protein synthesized per unit of DNA decreased. Protein was lost owing to a greater decrease in the rate of protein synthesis, as a result of the loss of RNA and a lowered RNA activity. When depleted chicks were re-fed the control diet, rapid growth was achieved by a doubling of the fractional synthesis rate by 2 days. Initially, this was a result of increased RNA activity; by 5 days, the RNA/DNA ratio also increased. There was no evidence of a decrease in the fractional degradation rate during re-feeding. These results indicate that dietary-protein depletion and repletion cause changes in breast-muscle protein mass primarily through changes in the rate of protein synthesis.     

Synthesis and turnover of plasma-membrane proteins and glycoproteins in a neuroblastoma cell line.

A kinetic analysis of the appearance of 14C-labelled proteins in the surface membranes isolated from exponentially growing neuroblastoma cells (N2a) showed that the total membrane proteins reached a steady-state specific radioactivity in 18-20 h. However, examination of individual protein bands resolved by sodium dodecyl sulphate-urea-polyacrylamide-gel electrophoresis illustrated that differences in the kinetics of specific surface-membrane proteins could be detected. Although most of the protein bands reached a steady-state specific radioactivity at a time similar to that for total membrane proteins, at least two bands (mol. wt. 180000 and 130000) attained the steady-state within 8-10 h. It was shown by the use of dual-labelling techniques that these two protein bands turned over in the surface membranes of neuroblastoma N2a cells at least 180 and 150% faster than the total membrane protein. These two proteins were glycosylated and located on the outer surface of the cells, since they were labelled with radioactive carbohydrates and readily removed by treatment of the intact neuroblastoma cell with proteinases.     

RNA turnover and protein synthesis in fish cells

Protein synthesis in fish has been previously correlated with RNA content. The present study investigates whether protein and RNA synthesis rates are similarly related. Protein and RNA synthesis rates were determined from ³H-phenylalanine and ³H-uridine incorporation, respectively, and expressed as % · day⁻¹ and half-lives, respectively. Three fibroblast cell lines were used: BF-2, RTP, CHSE 214, which are derived from the bluegill, rainbow trout and Chinook salmon, respectively. These cells contained similar RNA concentrations (∼175 μg RNA · mg⁻¹ cell protein). Therefore differences in protein synthesis rates, BF-2 (31.3 ± 1.8)>RTP (25.1 ± 1.7)>CHSE 214 (17.6 ± 1.1), were attributable to RNA translational efficiency. The most translationally efficient RNA (BF-2 cells), 1.8 mg protein synthesised · μg⁻¹ RNA · day⁻¹, corresponded to the lowest RNA half-life, 75.4 ± 6.4 h. Translationally efficient RNA was also energetically efficient with BF-2 cells exploiting the least costly route of nucleotide supply (i.e. exogenous salvage) 3.5–6.0 times more than the least translationally efficient RNA (CHSE 214 cells). These data suggest that differential nucleotide supply, between intracellular synthesis and exogenous salvage, constitutes the area of pre-translational flexibility exploited to maintain RNA synthesis as a fixed energetic cost component of protein synthesis. Accepted: 12 November 1999     

Proteasome-dependent turnover of protein disulfide isomerase in oxidatively stressed cells.

Generalized increases in protein oxidation and protein degradation in response to mild oxidative stress have been widely reported, but only a few individual proteins have actually been shown to undergo selective, oxidation-induced proteolysis. Our goal was to find such proteins in Clone 9 liver cells exposed to hydrogen peroxide. Using metabolic radiolabeling of intracellular proteins with [35S]cysteine/methionine, and analysis by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), we found at least three labeled proteins ("A," "B," and "C") whose levels were decreased significantly more than the generalized protein loss after mild oxidative stress. "Protein C" was excised from 2-D PAGE and subjected to N-terminal amino acid microsequencing. "Protein C" was identified as Protein Disulfide Isomerase or PDI (E.C. 5.3.4.1), and this identity was reconfirmed by Western blotting with a C-terminal anti-PDI monoclonal antibody. A combination of quantitative radiometry and Western blotting in 2-D PAGE revealed that PDI was selectively degraded and then new PDI was synthesized, following H2O2 exposure. PDI degradation was blocked by inhibitors of the proteasome, and by cell treatment with proteasome C2 subunit antisense oligonucleotides, indicating that the proteasome was largely responsible for oxidation-induced PDI degradation.     

The dynamics of the proteome: strategies for measuring protein turnover on a proteome-wide scale.

Quantitative proteomics captures the steady-state amount of a protein in a cell but does not explain how a change in protein amount is manifest -- whether through a change in synthesis or a change in degradation. If we are to understand the changes in the proteome, we will need to define such processes. In this brief review, strategies for the determination of intracellular protein dynamics on a proteome-wide scale are discussed.     

The effect of glutamine on protein turnover in chick skeletal muscle in vitro.

The effect of glutamine on the rates of protein synthesis and degradation was studied in isolated chick extensor digitorum communis muscles incubated in the presence of plasma concentrations of amino acids. Addition of 0.5-15 mM-glutamine increases (P less than 0.01) intracellular glutamine concentrations by 31-670%. There is a positive relationship (r = 0.975, P less than 0.01) between intracellular glutamine concentration and the rate of muscle protein synthesis measured by the incorporation of [3H]phenylalanine. The stimulating effect of 15 mM-glutamine on protein synthesis was decreased from 58 to 19% in muscles incubated in the absence of tyrosine. The rates of protein degradation, estimated from [3H]phenylalanine release from muscle proteins prelabelled in vivo, decreased (P less than 0.05) by 15-30% in the presence of 4-15 mM-glutamine when compared with muscles incubated in the presence of physiological concentrations of glutamine (0.5-1 mM). Glutamine concentrations ranging from 2 to 15 mM appear to have an overall anabolic effect on chick skeletal muscles incubated in vitro.     

Biosynthesis and turnover of DOPA-containing proteins by human cells

Protein-bound 3,4-dihydroxyphenylalanine (PB-DOPA) is a major product of hydroxyl radical attack on tyrosine residues of proteins. Levels of PB-DOPA in cells and tissues have been shown to be greatly elevated in age-related diseases. We demonstrate for the first time that l-DOPA (levodopa) can be biosynthetically incorporated into cell proteins by human cells (THP-1 monocytes and monocyte-derived macrophages). The DOPA-containing proteins generated were selectively visualized on PVDF membranes using a redox-cycling staining method. Many cell proteins contained DOPA and seemed to be synthesized as their full-length forms. The cellular removal of DOPA-containing proteins by THP-1 cells was by proteolysis involving both the proteasomal and the lysosomal systems. The rate of cellular proteolysis of DOPA-containing proteins increased at lower levels of DOPA incorporation but decreased at higher levels of DOPA incorporation. The decreased rate of degradation was accompanied by an increase in the activity of cathepsins B and L but the activity of cathepsin S increased only at lower levels of DOPA incorporation. These data raise the possibility that PB-DOPA could be generated in vivo from l-DOPA, which is the most widely used treatment for Parkinson disease.     

Dynamic turnover of arabinogalactan proteins in cultured Arabidopsis cells

Arabinogalactan proteins (AGPs) are very large proteoglycans thought to have more of a signaling than a structural role when secreted into the plant cell wall. AGPs are also the first known family of abundant plant proteins synthesized with glycosylphosphatidylinositol(GPI) anchors. Nascent cellular Arabidopsis AGPs, still bearing an intact GPI anchor, and AGPs copiously discharged into the culture medium after phospholipase-cleavage of their anchor were each represented by more than 15 seemingly homologous molecular species of increasing size. In washed cells ³H-ethanolamine was slowly incorporated into each AGP’s GPI anchor via phosphatidylethanolamine. Pulse labeling of AGPs by ³H-acetate and by ³H-galactose was much more rapid, allowing labeled AGP detection in the growth medium within 1 h. HPLC analysis of the radiolabel distribution in AGPs secreted within 1–8 h revealed a sharp preference for the larger molecular species. After several hours a population of smaller radioactive AGP species began to appear in the medium. Following certain manipulations of the cells newly secreted AGP species measured by HPLC on a relative mass basis formed a pattern surprisingly different from the radioactivity pattern, although larger species still dominated. Thus Arabidopsis cells appear capable of releasing higher mass AGP species apparently stored in cell wall sites along with a unique mixture of freshly synthesized AGPs in combinations potentially active in signaling.     

The effect of different antioxidants on glutathione turnover in human cell lines and their interaction with hydrogen peroxide

BACKGROUND: Glutathione plays crucial roles in antioxidant defence and glutathione deficiency contributes to oxidative stress and may therefore play a key role in the pathogenesis of many diseases. The objectives of the present study were to evaluate the effects on glutathione turnover of thiol and non-thiol antioxidants in human cell cultures and if any of the antioxidant had a short-term cellular effect against different levels of hydrogen peroxide. METHODS: We have investigated the effect on the total glutathione amount in HeLa and hepatoma cell cultures of thiol antioxidants in comparison with non-thiol antioxidants, such as a copper chelator, Vitamin C, and a flavonoid. Furthermore, we have investigated the short-term (within 24h) interaction of the different antioxidants with hydrogen peroxide. RESULTS AND CONCLUSION: Lipoic acid and quercetin (Quer) were the two antioxidants that showed the highest stimulation of glutathione synthesis in cell cultures as judged by the total glutathione amount. However, no antioxidant protected against hydrogen peroxide present in concentrations that lowered cell protein. This finding may be attributed to the fact that it is necessary to incubate cell cultures with antioxidants or small doses of oxidants for a period before the cultures are exposed to hydrogen peroxide in order to enhance the antioxidant defence. The presence of Quer and Vitamin C lowered cell protein and total glutathione even in cell cultures containing hydrogen peroxide in concentrations that did not lower cell protein. This finding might be attributed to pro-oxidant properties and formation of excess reactive oxygen species in the presence of Quer and Vitamin C.     

Significance of structural changes in proteins: expected errors in refined protein structures.

A quantitative expression key to evaluating significant structural differences or induced shifts between any two protein structures is derived. Because crystallography leads to reports of a single (or sometimes dual) position for each atom, the significance of any structural change based on comparison of two structures depends critically on knowing the expected precision of each median atomic position reported, and on extracting it for each atom, from the information provided in the Protein Data Bank and in the publication. The differences between structures of protein molecules that should be identical, and that are normally distributed, indicating that they are not affected by crystal contacts, were analyzed with respect to many potential indicators of structure precision, so as to extract, essentially by "machine learning" principles, a generally applicable expression involving the highest correlates. Eighteen refined crystal structures from the Protein Data Bank, in which there are multiple molecules in the crystallographic asymmetric unit, were selected and compared. The thermal B factor, the connectivity of the atom, and the ratio of the number of reflections to the number of atoms used in refinement correlate best with the magnitude of the positional differences between regions of the structures that otherwise would be expected to be the same. These results are embodied in a six-parameter equation that can be applied to any crystallographically refined structure to estimate the expected uncertainty in position of each atom. Structure change in a macromolecule can thus be referenced to the expected uncertainty in atomic position as reflected in the variance between otherwise identical structures with the observed values of correlated parameters.