Image correlation spectroscopy of randomly distributed disks

Image correlation spectroscopy (ICS) has been widely used to quantify spatiotemporal distributions of fluorescently labelled cell membrane proteins and receptors. When the membrane proteins are randomly distributed, ICS may be used to estimate protein densities, provided the proteins behave as point-like objects. At high protein area fraction, however, even randomly placed proteins cannot obey Poisson statistics, because of excluded area. The difficulty can arise if the protein effective area is quite large, or if proteins form large complexes or aggregate into clusters. In these cases, there is a need to determine the correct form of the intensity correlation function for hard disks in two dimensions, including the excluded area effects. We present an approximate but highly accurate algorithm for the computation of this correlation function. The correlation function was verified using test images of randomly distributed hard disks of uniform intensity convolved with the microscope point spread function. This algorithm can be readily modified to compute exact intensity correlation functions for any probe geometry, interaction potential, and fluorophore distribution; we show how to apply it to describe a random distribution of large proteins labeled with a single fluorophore.     

High divergence of reproductive tract proteins and their association with postzygotic reproductive isolation in Drosophila melanogaster and Drosophila virilis group species

The possible association between gonadal protein divergence and postzygotic reproductive isolation was investigated among species of the Drosophila melanogaster and D. virilis groups. Protein divergence was scored by high-resolution two-dimensional electrophoresis (2DE). Close to 500 protein spots from gonadal tissues (testis and ovary) and nongonadal tissues (malpighian tubules and brain) were analyzed and protein divergence was calculated based on presence vs absence. Both testis and ovary proteins showed higher divergence than nongonadal proteins, and also a highly significant positive correlation with postzygotic reproductive isolation but a weaker correlation with prezygotic reproductive isolation. Particularly, a positive and significant correlation was found between proteins expressed in the testis and postzygotic reproductive isolation among closely related species such as the within-phylad species in the D. virilis group. The high levels of male-reproductive-tract protein divergence between species might be associated with F₁ hybrid male sterility among closely related species. If so, a lower level of ovary protein divergence should be expected on the basis that F₁ female hybrids are fully fertile. However, this is not necessarily true if relatively few genes are responsible for the reproductive isolation observed between closely related species, as recent studies seem to suggest. We suggest that the faster rate of evolution of gonadal proteins in comparison to nongonadal proteins and the association of that rate with postzygotic reproductive isolation may be the result of episodic and/or sexual selection on male and female molecular traits. Correspondence to: A. Civetta     

Understanding the relationship between the primary structure of proteins and its propensity to be soluble on overexpression in Escherichia coli

Solubility of proteins on overexpression in Escherichia coli is a manifestation of the net effect of several sequence-dependent and sequence-independent factors. This study aims to delineate the relationship between the primary structure and solubility on overexpression. The amino acid sequences of proteins reported to be soluble or to form inclusion bodies on overexpression in E. coli under normal growth conditions were analyzed. The results show a positive correlation between thermostability and solubility of proteins, and an inverse correlation between the in vivo half-life of proteins and solubility. The amino acid (Asn, Thr, Tyr) composition and the tripeptide frequency of the protein were also found to influence its solubility on overexpression. The amino acids that were seen to be present in a comparatively higher frequency in inclusion body-forming proteins have a higher sheet propensity, whereas those that are seen more in soluble proteins have a higher helix propensity; this is indicative of a possible correlation between sheet propensity and inclusion body formation. Thus, the present analysis shows that thermostability, in vivo half-life, Asn, Thr, and Tyr content, and tripeptide composition of a protein are correlated to the propensity of a protein to be soluble on overexpression in E. coli. The precise mechanism by which these properties affect the solubility status of the overexpressed protein remains to be understood.     

Computational studies on mutant protein stability: The correlation between surface thermal expansion and protein stability

Thermal stability of mutant proteins has been investigated using temperature dependent molecular dynamics (MD) simulations in vacuo. The numerical modeling was aimed at mimicking protein expansion upon heating. After the conditions for an expanding protein accessible surface area were established for T4 lysozyme and barnase wild-type proteins, MD simulations were carried out under the same conditions using the crystal structures of several mutant proteins. The computed thermal expansion of the accessible surface area of mutant proteins was found to be strongly correlated with their experimentally measured stabilities. A similar, albeit weaker, correlation was observed for model mutant proteins. This opens the possibility of obtaining stability information directly from protein structure.     

A novel correlation for protein diffusion coefficients based on molecular weight and radius of gyration

A new correlation is proposed for the prediction of protein diffusion coefficients in free solution. Molecular weight and radius of gyration of proteins are employed as correlation parameters in this method. Both parameters can be easily found in the literature. The correlation works well for diverse proteins with different shapes and extensive molecular weight. Furthermore, this method does not require a preassumption regarding the protein shape while it offers a rapid and convenient calculation with a high accuracy. Also, the proposed correlation can elucidate the estimation deviation of previous correlation methods in the literature.     

A Sampling of the Yeast Proteome

In this study, we examined yeast proteins by two-dimensional (2D) gel electrophoresis and gathered quantitative information from about 1,400 spots. We found that there is an enormous range of protein abundance and, for identified spots, a good correlation between protein abundance, mRNA abundance, and codon bias. For each molecule of well-translated mRNA, there were about 4,000 molecules of protein. The relative abundance of proteins was measured in glucose and ethanol media. Protein turnover was examined and found to be insignificant for abundant proteins. Some phosphoproteins were identified. The behavior of proteins in differential centrifugation experiments was examined. Such experiments with 2D gels can give a global view of the yeast proteome.     

Discordant protein and mRNA expression in lung adenocarcinomas

The relationship between gene expression measured at the mRNA level and the corresponding protein level is not well characterized in human cancer. In this study, we compared mRNA and protein expression for a cohort of genes in the same lung adenocarcinomas. The abundance of 165 protein spots representing 98 individual genes was analyzed in 76 lung adenocarcinomas and nine non-neoplastic lung tissues using two-dimensional polyacrylamide gel electrophoresis. Specific polypeptides were identified using matrix-assisted laser desorption/ionization mass spectrometry. For the same 85 samples, mRNA levels were determined using oligonucleotide microarrays, allowing a comparative analysis of mRNA and protein expression among the 165 protein spots. Twenty-eight of the 165 protein spots (17%) or 21 of 98 genes (21.4%) had a statistically significant correlation between protein and mRNA expression (r > 0.2445; p < 0.05); however, among all 165 proteins the correlation coefficient values (r) ranged from -0.467 to 0.442. Correlation coefficient values were not related to protein abundance. Further, no significant correlation between mRNA and protein expression was found (r = -0.025) if the average levels of mRNA or protein among all samples were applied across the 165 protein spots (98 genes). The mRNA/protein correlation coefficient also varied among proteins with multiple isoforms, indicating potentially separate isoform-specific mechanisms for the regulation of protein abundance. Among the 21 genes with a significant correlation between mRNA and protein, five genes differed significantly between stage I and stage III lung adenocarcinomas. Using a quantitative analysis of mRNA and protein expression within the same lung adenocarcinomas, we showed that only a subset of the proteins exhibited a significant correlation with mRNA abundance.     

Estimation of diffusion coefficients of proteins

A correlation for estimating the diffusion coefficients of protein molecules is presented. The correlation is based upon literature values of the protein diffusion coefficients and molal volumes for 143 proteins. The correlation can be used for the estimation of diffusion coefficients using only molecular weight. Accuracy is such that a linear regression on 301 proteins showed 75% of the diffusion coefficients estimated fell within 20% of the experimental values. The relationship between this correlation, the Stokes–Einstein equation, and the Wilke–Chang correlation is discussed.     

Studies on the relationship between the degradative rates of proteins in vivo and their isoelectric points

Acidic proteins tend to be degraded more rapidly than neutral or basic proteins in rat liver, skeletal muscle, kidney and brain and in mouse liver and skeletal muscle. We now report a similar relationship among soluble proteins from rat lung, heart and testes, and from human fibroblasts and mouse-embryo cells grown in culture. These findings indicate that the correlation between protein net charge and degradative rate is a general characteristic of intracellular protein degradation in mammals. This relationship between isoelectric point and half-life appears to be distinct from the previously reported correlation between subunit molecular weight and protein half-lives. The more rapid degradation of acidic proteins does not result from their being of larger molecular weight than neutral or basic proteins. Furthermore, proteins within specific isoelectric point ranges still exhibit a relationship between subunit size and half-life. Finally, a group of membrane or organelle-associated proteins that are insoluble in phosphate-buffered saline and water but soluble in 1% Triton X-100 exhibit a correlation between size and half-life, but not between net charge and half-life. The biochemical reasons for the relationship between protein isoelectric point and half-life are unclear, although several possible explanations are presented. It is not due to a greater sensitivity of acidic proteins to proteolytic attack since experiments with a variety of endoproteinases, including trypsin, chymotrypsin, Pronase, papain, chymopapain, Staphylococcus aureus V8 proteinase, pepsin and lysosomal cathepsins from rat liver, have failed to demonstrate more rapid digestion of acidic proteins.     

Correlations of amino acids in proteins

A correlation analysis among 20 amino acids is performed for four protein structural classes (, β, /β, and +β) in a total of 204 proteins. The correlation relationships among amino acids can be classified into the following four types: (1) strong positive correlation, (2) strong negative correlation, (3) weak correlation, and (4) no correlation. The correlation relationships are different for different proteins and are correlated with the features of their structural classes. The amino acids with the weak correlation relationship can be treated as the independent basis functions for the space where proteins are defined. The amino acids with large correlation coefficients are linear correlative with each other and they are not independent. The strong correlation among amino acids reflects their mutual constrained relationship, as exhibited by their relevant structural features. The information obtained through the correlation analysis is used for predicting protein structural classes and a better prediction quality is obtained than that by the simple geometry distance methods without taking into account the correlation effects.     

Destabilizing mutations promote membrane protein misfolding

In this work, the relationship between stability and propensity to misfold was probed for a series of purified variants of the polytopic integral membrane protein diacylglycerol kinase. It was observed that there was a strong correlation between stability and folding efficiency. The most common mutations that promoted misfolding were those which also destabilized the protein. These results imply that by targeting unstable membrane proteins for degradation, cellular protein folding quality control can eliminate proteins that have a high intrinsic propensity to misfold into aberrant structures. Moreover, the more rare class of amino acid mutations that promote misfolding without perturbing stability may be particularly dangerous because the mutant proteins may evade the surveillance of cellular quality control systems.     

Proteomics-based identification of biomarkers for predicting sensitivity to a PI3-kinase inhibitor in cancer

To identify biomarkers for predicting sensitivity to phosphatidylinositol 3-kinase (PI3K) inhibitors, we have developed a proteomics-based approach. Using surface-enhanced laser desorption-ionization time-of-flight mass spectrometry (SELDI-TOF MS), we measured the expression of 393 proteins in 39 human cancer cell lines (JFCR-39), and combined it with our previously established chemosensitivity database to select for proteins whose expressions show significant correlations to drug sensitivities. This integrated approach allowed us to identify peaks from two proteins, 11.6 and 11.8 kDa, that showed significant correlations with the sensitivity to a PI3K inhibitor, LY294002. We found that the 11.8 kDa protein was a phosphorylated form of the 11.6 kDa protein. While the 11.8 kDa protein showed a positive correlation with the sensitivity to LY294002, the 11.6 kDa protein showed a negative correlation with that of the LY294002. The 11.6 kDa protein was purified chromatographically, and was identified by SELDI-TOF MS as the ribosomal P2 protein, which possesses two prospective phosphorylation sites. These results suggested that the phosphorylation status of the ribosomal P2 was responsible for determining the sensitivity to LY294002, and that the ribosomal P2 could be a potential biomarker for predicting chemosensitivity.     

Predicting Protein-Protein Interaction by the Mirrortree Method: Possibilities and Limitations

Molecular co-evolution analysis as a sequence-only based method has been used to predict protein-protein interactions. In co-evolution analysis, Pearson''s correlation within the mirrortree method is a well-known way of quantifying the correlation between protein pairs. Here we studied the mirrortree method on both known interacting protein pairs and sets of presumed non-interacting protein pairs, to evaluate the utility of this correlation analysis method for predicting protein-protein interactions within eukaryotes. We varied metrics for computing evolutionary distance and evolutionary span of the species analyzed. We found the differences between co-evolutionary correlation scores of the interacting and non-interacting proteins, normalized for evolutionary span, to be significantly predictive for proteins conserved over a wide range of eukaryotic clades (from mammals to fungi). On the other hand, for narrower ranges of evolutionary span, the predictive power was much weaker.     

Nanosecond segmental mobilities of tryptophan residues in proteins observed by lifetime-resolved fluorescence anisotropies.

Steady-state and lifetime-resolved fluorescence anisotropy measurements of protein fluorescence were used to investigate the depolarizing motions of tryptophan residues in proteins. Lifetime resolution was achieved by oxygen quenching. The proteins investigated were carbonic anhydrase, carboxypeptidase A, alpha-chymotrypsin, trypsin, pepsin, and bovine and human serum albumin. When corrected for overall protein rotation, the steady state anisotropies indicate that, on the average, the tryptophan residues in these proteins rotate 29 degrees +/- 6 degrees during the unquenched excited state lifetimes of these proteins, which range from 1.7 to 6.1 ns. The lifetime-resolved anisotropies reveal correlation times for these displacements ranging from 1 to 12 ns. On the average these correlation times are tenfold shorter than that expected for overall protein rotation. We conclude that the tryptophan residues in these proteins display remarkable freedom of motion within the protein matrix, which implies that these matrices are highly flexible on the nanosecond time scale.     

Comparison of protein expression lists from mass spectrometry of human blood fluids using exact peptide sequences versus BLAST

The proteins in blood were all first expressed as mRNAs from genes within cells. There are databases of human proteins that are known to be expressed as mRNA in human cells and tissues. Proteins identified from human blood by the correlation of mass spectra that fail to match human mRNA expression products may not be correct. We compared the proteins identified in human blood by mass spectrometry by 10 different groups by correlation to human and nonhuman nucleic acid sequences. We determined whether the peptides or proteins identified by the different groups mapped to the human known proteins of the Reference Sequence (RefSeq) database. We used Structured Query Language data base searches of the peptide sequences correlated to tandem mass spectrometry spectra and basic local alignment search tool analysis of the identified full length proteins to control for correlation to the wrong peptide sequence or the existence of the same or very similar peptide sequence shared by more than one protein. Mass spectra were correlated against large protein data bases that contain many sequences that may not be expressed in human beings yet the search returned a very high percentage of peptides or proteins that are known to be found in humans. Only about 5% of proteins mapped to hypothetical sequences, which is in agreement with the reported false-positive rate of searching algorithms conditions. The results were highly enriched in secreted and soluble proteins and diminished in insoluble or membrane proteins. Most of the proteins identified were relatively short and showed a similar size distribution compared to the RefSeq database. At least three groups agree on a nonredundant set of 1671 types of proteins and a nonredundant set of 3151 proteins were identified by at least three peptides.     

Immuno-qPCR detection of the tandem affinity purification (TAP)-tag as a sensitive and accurate tool suitable for large-scale protein quantification

The tandem affinity purification (TAP)-tag has rapidly gained a wide popularity, mostly in studies on protein interactions, but lately also in large-scale protein quantification studies. We have developed an immuno-quantitative real-time PCR (qPCR) method to achieve rapid, sensitive and accurate quantification of TAP-tagged (and protein A-tagged) proteins in yeast with a detection range between 10(7) and 10(10) molecules. The immuno-qPCR protein quantification showed an excellent correlation to the published in vivo fluorescent protein (GFP)-based large-scale protein quantifications, but allowed for a much higher sensitivity. The correlation with published data from the large-scale Western blotting-based quantification of the TAP-tag was lower, but the sensitivity of detection was on roughly the same level. The practical use of the immuno-qPCR approach was demonstrated by analysis of osmo-regulated proteins, where the 2000-fold increase in expression of Catalase (Ctt1p), from an extremely low basal expression, could be accurately quantified. All steps of the method, from cell growth, to protein extraction and determination and the immuno-qPCR reaction itself are potentially amenable to automatization. Therefore, since the TAP-tag and protein A are useful in most model organisms, the immuno-qPCR method is both generic and suitable for large-scale studies.