currents
M em brane Protein M aintenance One of the main challenges to proteome characterization is integral membrane proteins, the structures and activities of which are often dependent upon their lipid milieu. To get around this problem, researchers have relied on lipid or detergent micelles or bicelles to maintain such proteins, but these can be technically unwieldy and do not suit all analytical methods. Amphipols, amphipathic polymers with alternating polar and nonpolar sidechains, offer a smaller alternative to micelles and solubilize integral membrane proteins by forming a toroid around the transmembrane segments. One shortcoming of amphipols, however, is that their possible effects on enzyme activity have not been fully documented. To address this situation, Charles Sanders (now at Vanderbilt University, Nashville, TN) and colleagues at Case Western Reserve University (Cleveland) and Anatrace, Inc. (Maumee, OH) examined the activity of E. coli diacylglycerol kinase (DAGK) under various micellar and amphipolar conditions ( J. Am. Chem. Soc. 2002, 124, 11594–11595). The researchers added DAGK to a 0.5% solution of decyl maltoside (DM) micelles as well as solutions of the zwitterionic amphipol PMAL-B-100 at concentrations where there was some (0.5%) or no (0%) protein-free amphipol. They then compared the kinetic stability and activity of DAGK in the mixtures. After incubation at 37 °C for 48 h, the DAGK in the DM micelles retained 78% of its original activity, while the enzyme in 0.5% and 0% PMAL-B-100 retained 71% and 54% of its activ-
Prom oters and Gene Expression It has been assumed by many in the research community that comparative studies of large groups of gene promoter sequences will lead to a better understanding of promoter architecture, but this assumption might be too simplistic. Vaclav Mach, a researcher at the Czech Academy of Sciences (Ceske Budejovice), warns that the two prominent promoter resources— the Transcription Regulatory Regions Database and the Eukaryotic Promoter Database—rely solely on published data and therefore often neglect unpublished but experimentally characterized GenBank and EMBL entries. By the same token, he says, GenBank/EMBL queries for promoter information result © 2002 American Chemical Society
in a plethora of inadequately annotated information that requires extensive manual study. Furthermore, such a query cannot offer information about promoter activity and gene expression patterns. To address these problems, Mach developed the PRESTA (promoter EST association) algorithm that will link promoter information from GenBank/EMBL searches with expressed sequence tag (EST) data (Gen. Biol. 2002, 3, 0050.1–0050.7). Interactively accessible via the Internet(http://baloun. entu.cas.cz/presta), PRESTA evaluates entries using a context-dependent text analysis, parsing the entry into desired features such as “5⬘ tr”, “prim_ transcript”, “tata_signal”, and “promoter”, and screening out features such as “puta-
ity, respectively. Similarly, the activities of the PMAL-B-100 samples were 53 units/mg (0.5%) and 36 units/ mg (0%) as compared to 51 units/mg for DAGK in a mixed micelle of DM and the polar lipid cardiolipin W rapping up m em brane proteins.The amphi(CL). In the absence pathic polymers (amphipols) PMAL-B-100 and of CL, the activity in OAPA-20 give structural support to integral DM micelles fell to membrane proteins by forming a toroid around 1 unit/mg. As well, the their transmembrane segments. (Reproduced Mi c h a e l i s – Me n t o n with permission from Gorzelle, B. M.; et al. J. kinetics of the enzyme Am. Chem. Soc. 2002,124, 11594–11595.) in 0.5% PMAL-B-100 and DM/CL were almost identical. The researchers then examined the effect of the zwitterionic nature of the amphipol on DAGK activity. In PMAL-B-50, which is 50:50 anionic to zwitterionic, DAGK exhibited about half of the activity of the enzyme in PMAL-B-100, whereas in PMAL-B-0 and OAPA-20 (both of which are totally anionic) enzyme activity fell to 1 unit/mg. “The availability of at least one amphipol,” say the authors, “which can sustain the full functional activity of membrane proteins in the complete absence of detergents or lipids extends the range of potential applications of amphipols.”
tive”. These features are aggregated into collections that contain information about downstream transcription start sites. The algorithm then coordinates the promoter information with expression data by using the downstream sequences for high-stringency BLAST queries against an EST database. With the EST information, PRESTA can then confirm the promoterbased transcription start site. It can also compare sequences to eliminate duplicates using a directly incorporated SEQALN algorithm. To demonstrate the algorithm’s utility, Mach searched GenBank with the desired features mentioned above, which resulted in 2300 human and 1500 mouse entries. But when these results were screened with PRESTA, the number of
retained upstream promoter regions was reduced to 470 human and 280 mouse sequences, in an average processing time of 10 s. Similarly, a more complex query of both GenBank and EMBL resulted in 12,000 human and 5500 mouse entries that, when connected to EST data, were reduced to 552 and 241 entries, respectively, with an average length of 750 bases. To ensure that he didn’t miss promoters that were not linked to EST data, Mach screened the data using the promoter-predicting tools FirstEF, Promoter Inspector, and EpoNINE, but the results were inconclusive. As enthusiastic as he is, though, Mach warns that PRESTA results should only be seen as semiqualitative because of its reliance on published data, which is ob-
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currents viously skewed toward the systems most heavily studied. To some extent, this problem is offset by the sheer volume of ESTs available in the various databases—numbering in the millions. Regardless, Mach believes that PRESTA is one way to link structural and functional genetic information and should be seen as a complementary resource to other such databases and algorithms.
Conserved CellSignaling Cell communication is critical for the development and physiology of eukaryotic organisms, with signaling molecules typically taking one of two forms: membrane-permeable signals (e.g., steroids), or molecules transported by surface receptors (e.g., growth factors). Prokaryotes have likewise developed smallmolecule signals—often modified amino acids or proteolytically activated peptides—for intercellular communication. One example of a signal in eukaryotes is the epidermal growth factor (EGF) family and its affiliated receptors (EGFr) involved in cell fate and growth. Recently, Marco Gallio and colleagues at the Karolinska Institute and Soderstorns Hogskola (both in Huddinge, Sweden) and Case Western Reserve University (Cleveland) examined one member of the EGF family, the Drosophila transmembrane protein Rhomboid (RHO), and tried to determine whether it had functional partners in the prokaryotic world (Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 12208–12213). The researchers used the RHO sequence to search nonredundant databases for eukaryotic and prokaryotic RHO-related (RHOr) sequences. They were able to identify 50 microbial RHOr sequences, from which they generated a
phylogenetic tree. Settling on one particular sequence, the aarA gene of Providencia stuartii, a bacterium responsible for opportunistic infections in humans, the researchers determined whether the two proteins were functionally conservedthat is, whether AarA could replace RHO, and vice versa, in flies. Creating flies that overexpressed AarA, the researchers found that the transgenic flies had wing and eye morphologies similar to flies that overexpressed RHO. Correspondingly, reducing the dose of EGFr had the same effect whether the flies expressed AarA or RHO, and a dominant negative EGFr construct totally suppressed any AarAinduced effects, indicating that both AarA and RHO were working through the same mechanism. In comparison, AarA was able to correct the effects of rho mutations in flies. Finally, when one of the Drosophila RHO proteins was expressed in aarA mutant P. stuartii bacteria, the protein was able to rescue cell communication. The conservation of the two signaling proteins in or-
Signaling m echanism conservation? (a) The fly protein RHO cleaves the SPI peptide to propagate cell signaling. (b) Similarly, the bacterial protein AarA might cleave a peptide pheromone to initiate cell signaling. (Adapted with permission from Gallio, M.; et al. Proc. Natl. Acad. Sci. U.S.A. 2002,99, 12208– 12213. Copyright 2002 National Academy of Sciences, U.S.A.)
ganisms vastly separated on the evolutionary tree suggests that more signaling networks could have a common ancestry and opens the door to an expanded list of model systems for studying signaling defects.
M echanicalUnfolding
and the transition states (xu) were concurrently estimated. The rate constants at zeroforce were determined in this manner and used to calculate the degree of structure formation (the Φ value) in the transition states for the mutants. Specifically, Φ is the ratio of the mutant’s loss of wild-type-like stability in the transition state compared to the native state. Initial experiments yielded rate constants with extremely large errors of about 1 order of magnitude. This, it
Molecular dynamics simulations are generally required for detailed and quantitative descriptions of protein unfolding pathways. Experiments usually provide only qualitative characterizations of the energy landscape made from bulk molecular measurements using a chemical denaturant, as opposed to single-molecule unfolding analysis initiated by mechanical force in the simulations. To address this A tw o-state m odel unfolds.A protein will issue, Jane Clark and undergo several energy state changes (∅G) as it unfolds from its native (N) structure to a colleagues from the denatured (D) state via a transition state (‡). Medical Research (Adapted with permission from Best, R. B.; et Council Center for al. Proc. Natl. Acad. Sci. U.S.A. 2002,99, Protein Engineering 12143–12148. Copyright 2002 National (Cambridge, England) Academy of Sciences, U.S.A.) used atomic force was observed, was due to a microscopy (AFM) to very strong coupling between probe mechanical unfolding the fitted xu and ku values, pathways that will allow more where a slight error in xu redirect comparisons of the sulted in a very large one in experimental to the theoretical ku. But, if the values of xu of (Proc. Natl. Acad. Sci. U.S.A. the mutant and wild-type 2002, 99, 12143–12148). proteins are within error of The researchers applied a one another, then it is a reastrategy comparing the dynasonable assumption to use mics of mutated and wildthe average value as a contype proteins to probe the stant xu. By using this apimportance of interactions proach, rate constants and in different parts of the strucsubsequent Φ values were ture along the folding/unobtained with much smaller folding pathway. They did errors. this with AFM force spectra Furthermore, it was posof the wild-type muscular sible to calculate reasonable protein titin 127 and two modresults for Φ by direct comerate mutations (i.e., unlikely parisons of the unfolding to change the final protein forces without the need for structure). The unfolding rate Monte Carlo statistics. constants (ku) were estimated at each applied force by The actual results were a using Monte Carlo simulaΦ of about 1.0 for the protein tions in which the lengths of with a mutation at residue 60, molecule that unfolded indicating complete native between the native proteins interactions for this residue in
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currents the transition state, and a Φ of about 0.6 for the protein mutated at residue 13, indicating only a fraction of native contacts in the transition state. A full Φ analysis of the mechanical unfolding path way of titin 127 will be published separately.
Library Screening w ith M S
mixture showed several noncovalently bound species. The complexes of interest were then isolated using the stored-waveform inverse FT technique and broken into their constituent parts using infrared multiphoton laser dissociation. The two main peptide species were identified as pYEDI/pYEEV and pYEEI, the latter of which is the high-affinity ligand for the Src SH2 domain.
The applications of MS in proteomics and drug discovery are rapidly expanding, and FTICR MS offers the added benefit of being able to identify M ultiple binding partners.The FT-ICR MS spectrum of noncovalent Hck SH2 domain bound noncovalently to various ligands complexes from a peptide library with the consensus Ac-GpYEXXusing “soft” Eda. (Adapted with permission from Wigger, M.; et al. J. ionization Am. Soc. Mass Spectrom. 2002,13, 1162–1169. Copyright techniques 2002 Elsevier Science.) such as ESI The researchers then perand MALDI. In some cases, formed a similar high-affinitythe gas-phase techniques can ligand isolation by passing the offer results as good as those peptide library over a column achieved with liquid-phase of Hck SH2 domain bound to experiments. For example, agarose beads, washing the Steven Benner and colleagues column with ammonium at the University of Florida acetate, and eluting the pep(Gainesville) and the tides with formic acid. The National High Magnetic Field isolated peptides were then Laboratory (Tallahassee, FL) sequenced. In this case, the recently combined ESI-FTpreferred peptides were pYE ICR MS with other techniques followed by two hydrophobic to isolate and identify pro(φ) residues (pYEφφ) and pYE tein-binding ligands from a followed by D or E and I or V, combinatorial library of pepthe latter of which matches tides and compared the the peptides isolated using results with those using proMS (i.e., pYEDI/pYEEV). tein bound to agarose resin The researchers ex( J. Am. Soc. Mass Spectrom. plained that they could not 2002, 13, 1162–1169). isolate the pYEφφ peptide The researchers screened using the MS-based method a 324-member library combecause of prising peptides with the conthe predominance of polar sensus sequence Ac-GpYEXXinteractions over hydrophoEda (where pY is phosphotybic interactions in the gas rosine, X is any amino acid phase and said that this is except C or L, and Eda is ethone shortcoming of their apylenediamine) against the Src proach. Even given this diffihomology 2 (SH2) domain of culty, the authors believe that the hematopoietic cell kinase the speed and simplicity of (Hck), a protein involved in their MS noncovalent bindB-cell function. ESI-FT-ICR ing assay makes it preferable MS of the peptide–protein 494
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to solution-based methods for efficient screening of large combinatorial libraries.
Changing Channels Traditional ion channel studies have relied on patch clamps to detect changes in the voltage potential of the cell as the channel opens and closes. Ion channel dynamics, however, are subtler because the relative positions of the residues can change without the opening or closing of the channel. To address this problem, researchers have fluorescently tagged the channel proteins and used the change in fluorescence (∅F) caused by the change in the fluorophore’s environment as a signal of protein alteration. But even these studies have been confined to large ensembles of proteins where variations from protein to protein can blur distinct transitions. Thus, Ehud Isacoff and colleagues at the Hamamatsu University School of Medicine (Japan) and the Lawrence Berkeley National Laboratory (CA) attempted to follow the ∅F of individual ion channels on the surface of a Xenopus oocyte (Proc. Natl. Acad. Sci. U.S.A. 2002, 99,
tetramethylrhodamine-5maleimide (TMRM). The positions were chosen because their ∅F occurs in opposite directions upon cell depolarization and they thereby serve as internal controls for one another. Because the excited-state lifetime of TMRM is on the nanosecond scale—as opposed to the typical microsecond scale—it is an ideal reporter of subtle transitions. The researchers examined oocytes using total internal reflection (TIR) fluorescence microscopy, exposing the oocytes to an Nd-YAG laser, which established an evanescent field of excitation and caused the TMRM on individual channels to fluoresce. When voltage clamps were attached to the oocytes, the researchers were able to detect ∅F on a CCD camera by subtracting the signals from individual image pixels acquired before depolarization from those acquired during depolarization. In contrast to fluorophores in solution, the researchers found that the signals from the tagged, membrane-bound proteins were
A difference offluorescence.In the transition from depolarized (A) to repolarized (B), fluorescently tagged ion channels undergo structural rearrangements that are reflected in changes in their fluorescent signals (C). (Adapted with permission from Sonnleitner, A.; et al. Proc. Natl. Acad. Sci. U.S.A. 2002,99, 12759–12764. Copyright 2002 National Academy of Sciences, U.S.A.)
12759–12764). The researchers injected the cRNA that encodes a mutated version of the Shaker potassium channel into the frog oocytes in such a way that the resulting channels carried a single cys teine residue at either position 351 or 359, near the voltage–sensing S4 transmembrane segment.These cysteine residues served as attachment points for the fluorescent tag,
quite stable, with little or no lateral motion during signal capture. Although there were some problems with fluorescent signals from free TMRM bound nonspecifically to the oocyte surface and with bleaching of the signals due to photodestruction, the researchers were enthusiastic about extending their method to related structural rearrangements in other proteins or using fluo-
