currents
Crystallizing M em brane Proteins Within the vast number of protein structures in the PDB, only around 70 entries describe the structure of integral membrane proteins (IMPs), and less than half of these have an independent fold. Crystallization of any protein requires extensive purification, but in the case of IMPs, the isolation process can lead to lipid depletion, which can have destructive effects. Previous studies have shown that lipids can have structural or functional roles with the IMPs, and it has been suggested that in a detergent micelle devoid of lipid, IMP side chain and backbone bonds have more conformational freedom and might be more labile from increased water accessibility. Both factors complicate the crystallization process. To address this issue, William Cramer and colleagues at Purdue University (www.purdue. edu) recently proposed that small amounts of pure lipid might stabilize the protein−micelle complex (Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 5160–5163). The researchers chose to study the hetero-oligomeric cytochrome b6 f complex, an obligate dimer involved in generating a transmembrane proton electrochemical gradient for ATP synthesis.
Fluorescing for Phosphatases Protein phosphatases (PP) are vital components in intracellular signaling processes, and research has implicated the deregulation of some of these enzymes in various diseases. Current PP assay methods often involve measurements that do not allow for the continuous monitoring of enzyme activity and can be vulnerable to high background signals in crude cell extracts. But in a recent study, Anthony Cass and colleagues at Imperial College (www.ic.ac.uk) and the Novartis Institute for Medical Sciences (www.nibr.novar tis.com) described new fluorescent peptide probes that they say are suitable for more selective, “mix-and-read” high-throughput assays for the enzymes PP1 and PP2A (Anal. Chem. 2003, 75, 2042–2047). © 2003 American Chemical Society
Lipid-m ediated order.A schematic of the cytochrome b6f complex in a (A) membrane bilayer, (B) detergent micelle, and (C) detergent−lipid mixture. Researchers believe that the addition of lipid might promote a more ordered structure and thereby facilitate complex crystallization. (Adapted with permission from Zhang, H.; et al. Proc. Natl. Acad. Sci. U.S.A. 2003,100, 5160–5163. Copyright 2003, National Academy of Sciences, U.S.A.)
The researchers developed the probes on the basis of a fluorophore label that is sensitive to its chemical surroundings in such a way that the dephosphorylation of an adjacent phosphotheonine residue quenches its fluorescence. Such environmentally sensitive labels, which allow independent variation in signal generation and substrate specificity, have been applied in the past to monitor conformational changes and ligandbinding events in various proteins. Cass and his colleagues started out by synthesizing a peptide derived from a sequence surrounding threonine-97 from bovine myelin basic protein (a known kinase substrate) and preparing the corresponding phosphopeptide. They substituted the arginine residue adjacent to
The researchers used a detergent extraction to purify the 217-kDa complex from the cyanobacterium Mastigocladus laminosus and found that the purified complex contained less than 0.5 molecules of monogalactosyldiacyl glycerol per monomer. This contrasts sharply with the findings for previously studied complexes, which typically carry five lipid molecules per monomer. The addition of dioleolylphosphatidylcholine (DOPC), however, resulted in rapid cystallization, and the researchers were able to achieve 3.4-Å resolution using synchrotron radiation, which compared favorably with previous attempts that achieved only 12-Å resolution. The researchers found that lipid with a phosphatidylglycerol head group, which is native to M. laminosus, could be substituted for the non-native phosphatidylcholine head group. This finding suggested that “the augmented lipid is not bound specifically within the protein complex, but more likely in the protein−detergent boundary layer.” The researchers believe that their method might be of general use in the crystallization of IMPs, opening the door for a rapid expansion in the number of IMPs listed in the PDB.
the phosphotheonine with a cysteine to gain site-selective labeling ability and then attached various dyes to the phosphopeptide. The team then measured the flourescence signal for each upon the addition of PP1 or PP2A. The conjugate containing the fluorophore 7-fluorobenz-2-oxa1,3-diazole-4-sulfonamide (ABD-F) showed the largest enzyme-generated decrease in fluorescence intensity. The researchers measured the ABD-F intensity decrease continuously, and the resulting emission profile, when compared to the ABD-F-labeled unphosphorylated peptide, indicated a 56% quench in fluorescence upon complete dephosphorylation. They also showed that there was good correlation between the emission profile and the release of inorganic phosphate.
The team then synthesized derivatives of the phosphopeptide to find optimum substrates and found two analogues that significantly increased the enzymatic rates of PP1 and PP2A, respectively. “The faster turnover rates for the optimized phosphopeptide substrates,” they wrote, “make this assay suitable for high-density microplate assays.” The scientists used the PP1 and PP2A optimum substrates for 96-well IC50 assays of three natural PP inhibitors. The results showed strong agreement with literature IC50 values, providing evidence that these synthetic phosphopeptides accurately reflect physiological substrates. Experiments with PP2B and alkaline phosphatase indicated “a degree of specificity that could allow [the
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currents phosphopeptides] to be used in crude cell extracts for profiling protein phosphatase activities.”
Glycoprotein Production Protein-based therapeutics comprise almost 500 candidates in current clinical trials. Although many of these proteins can be produced inexpensively in bacteria, many others require complex posttranslational modifications to function correctly. These latter proteins are often produced in mammalian cell cultures, but such systems suffer from many drawbacks, including low yield, high cost, and potential retroviral contamination. Fungal protein-expression systems, by contrast, do not suffer from those problems and often yield protein titers of 15–30 g/L; however, they synthesize nonhuman N-glycans that are immunogenic in humans. Although many of the initial steps of protein Nglycosylation are common to fungi and mammals, the synthetic pathways diverge significantly when the glycoproteins
are exported to the Golgi. Thus, to successfully produce humanized glycoproteins in fungi, it would be necessary to humanize the fungal glycosylation machinery. Toward this end, Tillman Gerngross and colleagues at Dartmouth College (www.dartmouth.edu) recently created such a system in the yeast Pichia pastoris, using a combinatorial genetic library of protein fusions and a high-throughput screen (Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 5022–5027). Initially, the researchers deleted the gene (OCH1) for the endogenous yeast α-1,2mannosyltransferase, which initiates the outer-chain elongation in the early Golgi, and confirmed their results with PCR and Western blotting. They then followed the effects of their modifications by monitoring the glycosylation states of a secreted test protein using MALDI-TOF MS, finding that there was a noticeable shift to shorter glycans in the och1 strain. The researchers then created three gene libraries. The
ER (human, P. pastoris)
Man8
Golgi (human)
Golgi (P. pastoris)
Mns IA, IB and IC
1,6 MnT (Och1p)
Man5
Man9
GnTI
GlcNAcMan5
Complex Glycans
1,2 MnTs 1,6 MnTs b-1,N-GlcNAc b-1,4-GlcNAc b-1,2-GlcNAc b-1,4-Man a-1,6-Man a-1,2-Man a-1,3-Man
Man9-14
Neitherm an noryeast.Although the early steps of protein N-glycosylation are similar between humans and yeast, the two pathways diverge in the Golgi. (Adapted with permission from Choi, B.-K.; et al. Proc. Natl. Acad. Sci. U.S.A. 2003,100, 5022–5027. Copyright 2003, National Academy of Sciences, U.S.A.)
first library comprised sequences encoding N-terminal peptides of known fungal transmembrane proteins that localize to the endoplasmic reticulum or Golgi. The second library was composed of sequences encoding the catalytic domains of ␣-1,2-mannosidases from various organisms, which were chosen to cover a wide range of pH and temperature optima. And the final library comprised the catalytic domains from various 1,2-N-acetylglucosaminyltransferase I (GnTI) genes. The researchers then recombined the signal peptide library with the catalytic libraries to form a library of peptide−enzyme fusions that were tested in the och1 yeast. Several clones from the mannosidase fusion library produced humanized N-glycans intracellularly. The N-glycans could be further humanized in cells that also contained GnTI, although this step also required the co-transformation of the cells with a transporter protein for UDP-GlcNAc, a required substrate. According to the researchers, “This is a demonstration of a high-level hybrid N-glycan modification of a secreted protein in yeast and represents a significant step toward the ability to express fully human glycoproteins in yeast.”
Fuzzy M atching Gel electrophoresis imaging enables researchers to compare protein expression in samples taken from different sources, such as healthy and diseased tissues. Most of the software currently available for analyzing electrophoresis images requires intensive user interaction, but automated image comparison would expedite high-throughput studies and enable standardized, repeatable comparisons. Researchers from the University of Silesia (www.us.edu.pl) and Unilever R&D (www.uni
lever.com) have developed just such an automated matching method on the basis of the “fuzzy alignment” of features extracted from gel images (J. Chem. Inf. Comput. Sci. 2003, 43, 978–986). The authors used grid coordinates, areas, and intensities for control points or “landmarks” in reference images to align corresponding features in real and simulated 2-D gel images. Previously, landmarks had to be selected manually by an experienced user to avoid accidental similarities between noncorresponding points. This problem was addressed using “fuzzy matching” of image features, in which a circle is generated that covers 25% of the spots under consideration. The circle shrinks as noncorresponding spots are eliminated from consideration and the fit improves. Ideally, the circle shrinks until the features correspond on a oneto-one basis. Spots and landmarks that corresponded between the test and reference images were used to calculate the parameters for a global transformation grid, and localized transformations were adjusted for distortions that did not cover the entire image, improving the fit. The researchers interpolated intensity values for test points from nearest-neighbor grid points, and then they superimposed false-color versions of the image pairs for ease of comparison. When red and blue components were taken from one image and green components from another, features that did not occur in both images appeared as magenta or green. Where features coincided, the composite image appeared gray to black, depending on the intensity of the spots. Overall, the calculation was relatively fast, depending on the number of extracted features, but comparing a
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currents large number of features was not necessary to achieve a good match. The scientists obtained the best results by using local linear transformations, followed by a 16point intensity interpolation, but other combinations of grid transformations and intensity interpolations produced results that were almost as good. The method of intensity interpolation had the greatest effect on the quality of the fit.
Extracting M ALDIResults Sample preparation is critical to the success of MALDI-MS analysis. Although the drieddroplet method has been developed and refined over the past decade into a robust technique, it is not without its shortcomings. Because the method produces heterogeneous matrix−analyte deposits, it can be necessary to move the laser across the sample to find analyte-rich positions. Furthermore, when used for low-femtomole detection studies, the method suffers from reduced ion yields and poor detection efficiency of post-translationally modified peptides. To address these limitations, Sven Kjellström and Ole Nørregaard Jensen of the University of Southern Denmark (www.sdu. dk/Nat/bmb) recently developed a liquid−liquid extraction (LLE) system for the partitioning of hydrophobic and hydrophilic peptides in situ on the MALDI plate (Anal. Chem. 2003, 75, 2362–2369). To perform LLE, the researchers place a droplet of peptides in aqueous buffer onto the MALDI plate and then add a second droplet containing matrix in waterimmiscible organic solvent. The hydrophobic peptides move from the aqueous to the organic phase, the efficiency of which is affected by factors such as amino acid sequence, the pH and ionic strength of
the aqueous phase, researchers blotted and the nature of the the libraries onto solvent. Once the nitrocellulose and organic−matrix phase probed the memdries and crystallizes, brane with autolothe researchers move gous patient serum. the remaining aqueThey were able to ous material to a new identify 27 unique spot on the MALDI reactive clones from plate, where the the two libraries. process is either terThe researchers minated with the then performed a addition of matrix or second immunorepeated. screening of the 27 The researchers clones by probing initially tested their them with sera from system with a mix20 healthy volunOforganic extraction.Researchers combine peptides in aqueous buffer (A) with a water-immiscible organic solture of 3- to 30-kDa teers, 16 patients vent containing matrix (B) to extract hydrophobic peptides proteins, finding that with hepatitis-related (C). Once the organic phase dries and crystallizes (F1), the the hydrophobic prochronic liver injury, researchers remove the aqueous material (D) to a new teins extracted more and 20 patients with spot on the MALDI plate (E, F2). (Adapted with permission efficiently into HCC. Of the 27 from Kjellström, S.; Jensen, O. N. Anal. Chem. 2003,75, organic solvent plus clones, 6 reacted with 2362–2369.) matrix as opposed to autologous sera only, organic solvent 17 reacted with biomolecules, including memalone. Also, multiple rounds of healthy and HCC patient sera, brane proteins, in the future. LLE were often required to and 1 reacted with HCC and obtain the best results. The hepatitis patient sera. Alteam then performed LLE on Targeting Cancer though sera from six HCC a tryptic digest of phosphoryHepatocellular carcinoma patients did not react with the lated β-casein and found that (HCC) is a leading cause of remaining three clones, 70% of the more hydrophobic pepdeath in Asia and is often the the HCC samples reacted with tides partitioned into the terminal complication of liver at least one of the three. organic phase while the phosdiseases such as chronic The researchers identified phopeptides remained and inflammation and fibrosis. the three clones as Tat-bindwere effectively concentrated Although several therapeutic ing protein-1, or TBP-1; 4 in the aqueous phase. pathways have been recently integrin-binding protein, or The scientists then perdeveloped, the prognosis for p27(BBP); and ribosomal proformed extraction on a tryptic HCC is still quite poor. Vactein L30, or rpL30. Scientists digest of bovine serum albucine programs against tumororiginally described TBP-1 as min spiked with a phosphospecific peptides, however, a transcription factor of HIVpeptide, glycopeptide, and have started to show some 1. It also inhibits the replicaacetylated peptide to examine promise with various maligtion of hepatitis B virus. Previwhether LLE would effectively nant conditions. Recently, ous research has shown that partition other post-translaMasayuki Uemura and colp27(BBP) is overexpressed in tionally modified peptides. As leagues at Okayama Univerhuman colorectal cancer and they had hoped, they detected sity (www.okayama-u.ac.jp) rpL30 is overexpressed in the phosphopeptide and glyand Okayama Citizens’ Hospiprostate carcinoma. Even copeptide in the aqueous tal identified antigens that though each of the proteins is phase and the acetylated pepwere unique to HCC and that expressed constitutively in tide in the organic phase. The might serve as potential tarboth healthy and diseased researchers also determined gets for anti-HCC vaccines liver tissue, the researchers are that after an extensive wash(Cancer 2003, 97, 2474−2479). confident that they have idening procedure prior to in-gel The researchers created tified three HCC biomarkers. digestions, LLE was also effeccDNA libraries from two HCC They conclude, “Elucidative for the MALDI-MS analytumor samples, generating tion of the mechanism of this sis of protein bands from almost 106 clones. They then cancer-specific antigen proSDS-PAGE and 2DE. packaged the cDNA fragments duction might make it possiThe team plans to develop into phage vectors and transble to use these antigens in the technique and expand it to fected E. coli. After inducing the future for immune therapy analyzing other hydrophobic protein expression, the in patients with HCC.”
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