Simulating Protein Folding - American Chemical Society

may differ according to the sample content and concentration, buffer conduc- tivity and vis- cosity, capil- lary length and conditioning, and machine ...
2 downloads 0 Views 3MB Size

Compared to 2-DE, capillary electrophoresis (CE) offers shorter protein analysis times, requires smaller samples and fewer reagents, and is readily coupled to MS. Unfortunately, CE is hampered by problems of protein adsorption to the capillary wall and low sensitivity. Mari Tabuchi and Yoshinobu Baba of the University of Tokushima (Japan) recently addressed the first problem with the use of curdlan, a buffer additive that disrupts protein−capillary wall interactions. The second problem, however, has typically required that samples be concentrated using a modified sample injection method. This method, unfortunately, requires longer sampling capillaries, which remove the benefit of speed. To get around this problem, Tabuchi and Baba developed a new injection method that uses pressure to concentrate the sample while still using a standard-size capillary (Electrophoresis 2002, 23, 1138−1145). Using a commercial peptide mixture, the researchers compared the peak resolution and run times of a standard separation without pressure to similar separations using various pressure conditions. Tabuchi and Baba found that peak migration times were reduced when 10 mbar of pressure was applied to a sample upon injection and that peak shape was not significantly altered. Migration time was further reduced when the capillary outlet was free of buffer at the time of pressurization. This latter result, the researchers explained, was due to the freedom of the buffer, and therefore the sample, to move within the capillary when pressurized. The researchers then tried to optimize the system by using various pressures for varying lengths of time. The results indicated that a 10-mbar pulse of 8 s duration offered optimal

Chem icalM odifications and M S The historically accepted topology of the glycine receptor (GlyR), a major inhibitory neurotransmitter receptor in the mammalian spinal cord and lower brain, includes a large N-terminal domain tethered to four transmembrane α-helices, denoted M1–M4. However, recent experimental results have not supported the presence of four such αhelices, and some data have specifically pointed to M1 as, potentially, not being entirely α-helical. These findings have generated interest in refining the structural understanding of the GlyR receptor. To further examine GlyR topology, John Leite and Michael Cascio of the Uni© 2002 American Chemical Society

versity of Pittsburgh chemically modified the protein and performed mass spectral analysis (Biochemistry 2002, 41, 6140–6148). Specifically, they used tetranitromethane (TNM) to selectively nitrate tyrosine residues. They followed this with proteolytic M220 G221 L224 I225 Y228 I229 S231 L232 V235 I236 W239 I240 F242 W243

Y222 Y223 Q226 M227 P230 L233 I234 L237 S238 S241 I244 N245


M odified tyrosines.GlyR residues 220−246 are modeled as a transmembrane α-helix, and tyrosine (Y) residues modified by the tetranitromethane are shown as gray beads. (Adapted from Biochemistry 2002,41, 6140−6148.)

peak resolution, as higher pressures (50 mbar) or shorter times (2 s) led to peak broadening. Furthermore, the researchers found that the addition of curdlan to the buffer at optimal pressure and time conditions maintained the migration time while improving peak resolution. The researchers warn, however, that the optimal conditions may differ according to the sample content and concentration, buffer conducMethod I: 250 5 3 A sample injection tivity and vis1 4 (outlet: buffer) without pressurization cosity, capil6 7 2 8 lary length and 9 0 conditioning, 0 1 2 3 4 5 6 7 8 9 10 and machine 200 5 Method II: specifications. B 3 sample injection (outlet: buffer) 1 4 Tabuchi and 7 water pressurization 6 2 (outlet: buffer) 8 Baba suggest 9 that the new 0 0 1 2 3 4 5 6 7 8 9 10 methods will enable the use Method III: 150 3 5 C sample injection 4 of CE for high(outlet: no buffer) 1 7 water pressurization 8 throughput 2 6 (outlet: buffer) 9 applications. Absorption (mAV)

Optim izing CE Analysis

0 0









9 10

Perform ing w ellunderpressure.When capillary electrophoresis sample injection is followed by a burst of pressure, the sample peptides elute more quickly (Method II) than without the pressure (Method I). This effect is further enhanced by eliminating the buffer from the outlet before sample injection (Method III). (Reproduced with permission from Electrophoresis 2002,23, 1138−1145. Copyright 2002 John Wiley & Sons Ltd.)

digestion and MS, in which mass-shifted (+45 Da) spectra signals indicated tyrosine nitration. Because TNM only reacts with solvent-accessible tyrosine, the spectra reveal the receptor’s topology. By running TNM-treated samples in parallel to untreated samples through the mass spectrometer, Leite and Cascio identified 6 of the 16 GlyR tyrosine residues as being modified to o-nitrotyrosine. Four of these residues were located within the N-terminal domain of the protein. The researchers took advantage of a recent highresolution structure attained for an acetylcholine binding protein (AchBP), which carried a sequence highly homologous to the GlyR

N-terminal domain, to evaluate the quality of the tyrosine accessibility method. Sequence alignments between the two proteins showed that the four AchBP residues comparable to the modified GlyR tyrosines were solventaccessible, while others in this region were not, corroborating the modification results. The remaining two modified residues were located within the putative transmembrane M1 region. But if M1 did fold into a pure α-helix, the two tyrosines would be well within the interior of the lipid bilayer, precluding them from TNM modification. The modification results thus suggest that M1 does not, in fact, fold in

JournalofProteom e Research • Vol. 1, No. 4, 2002 299

currents this manner. The authors propose that the region may be composed of multiple β-sheets or, alternatively, it could consist of “a novel, unexpected secondary structure.” They are currently using cysteine substitution mutagenesis coupled to spin labeling techniques to further define GlyR M1 topology.

Sim ulating Protein Folding Many human diseases are the result of genetic mutations that lead to errors in protein folding, that is, the protein is produced but is unable to perform its normal function (e.g., sickle cell anemia and hemoglobin). Thus, understanding which internal and external factors influence the way a protein folds to its native state is a pressing issue. Unfortunately, it is difficult to study how a protein moves through its transition-state ensemble (TSE) from unfolded to native structure by conventional techniques such as mutagenesis or chemical cross-linking because the TSE is unstable, transient, and high-energy. Computational modeling using algorithms, such as molecular dynamics (MD) or Monte Carlo (MC) simulations, however, may provide clues to understanding the TSE. Previous MD and MC simulations of protein folding have identified transition-state structures that are consistent with experimental data. Recently, Jörg Gsponer and Amedea Caflisch of the University of Zurich (Switzerland) compared experimental folding data to an MD simulation of the src SH3 domain TSE (Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6719−6724). The SH3 domain is an all-β-structure formed by the orthogonal sandwiching of a β-hairpin (formed by the terminal segments) and a three-

stranded antiparallel β-sheet. Using the program CHARMM, the researchers modeled the protein by considering all of its heavy atoms and hydrogen atoms bound to nitrogen or oxygen. Contacts between residues were considered to exist when side chain-heavy atoms of two nonadjacent residues were within 6 Å. Nonpolar residues were predominantly selected because mutations of these residues typically have the greatest effect on overall folding. In agreement with experimental results, the simulation indicated that the protein termini move through several non-native positions during folding while the antiparallel β-sheet is largely folded (see figure). The simulation also indicated that several residue side chains form contacts with other side chains that are not found in the native structure. These non-native interactions

Folding through the transitionstate. Steps in one SH3 folding pathway. (Adapted with permission from Proc. Natl. Acad. Sci. U.S.A. 2002,99, 6719−6724.Copyright 2002 National Academy of Sciences U.S.A.)

occur predominantly in the turn regions and, in the case of two loops, must return to

nativelike packing before fast folding can occur. Taken together, these results demonstrate that an atomic-resolution description of the TSE can be developed from MD simulations.

Directand Sensitive Im m unodetection To perform immunodetection of proteins separated on a PAGE gel, the proteins must be transferred to membranes (a Western blot), because PAGE gels are impenetrable to antibodies. However, the efficiency of this transfer is variable, leading to potentially significant disparities between the electrophoresis results and the patterns and amounts of material that are immunoprobed. Agarose gels, by contrast, are permeable to antibodies. Thus, Gary Smejkal and his colleagues at Cleveland State University (Ohio) developed a method for directly immunoprobing proteins following agarose-gel electrophoresis, without the need for blots. In addition, they used chemiluminescent immunodetection as a sensitive alternative to immunostaining (Electrophoresis 2002, 23, 979−984). Initially, the researchers separated fibrinogen and its derivatives on standard 1.5-mm-thick agarose gels, which were then incubated with horseradish peroxidase (HRP)-conjugated rabbit antihuman fibrinogen antibodies. Subsequently, gels were either treated with luminolbased chemiluminescent substrates or stained with 3,3′-diaminobenzidene (DAB). Although chemiluminescent labeling achieved a detection sensitivity of 500 pg of fibrinogen per band,

DAB staining only detected 5 ng/band. In addition, certain fibrinogen oligomers and plasma fibrinogen multimers that were not detectable with DAB were easily observed by chemiluminescence. Although the agarose gel method eliminates the risk of misrepresenting electrophoretic separations due to blotting, other complications arise that generally do not occur with Western blot analysis. The greater thickness of the agarose gels makes complete washing of unbound antibodies much more difficult than it is for blots, where immunoprobing is primarily limited to the membrane surface. This problem resulted in notable background signals. In addition, the probe time and the amount of antibody that were needed were significantly greater. Thus, using a specialized frame, the researchers compressed the agarose gel (derivatized with 1.2% glyoxyl) down to a thickness of 0.3 mm. Because this compression was performed prior to immunoprobing and after electrophoresis, no background chemiluminescence or staining was observed and less time and material were necessary. Using the compressed gels, the relative sensitivity of chemiluminescence remained at about 10 times greater than for staining; however, both approaches were about 30 times less sensitive than the thick gel analysis. The researchers indicated that this was partly due to the lower HRP content of the antibody used in this part of the experiment. They pointed out, as well, that because no background noise was observed, the sensitivity could be improved by adding more antibody and more chemiluminescent substrates.

JournalofProteom e Research • Vol. 1, No. 4, 2002 301

currents Rapid Virus Identification Using M S Various mass spectrometric techniques have been used in the past to characterize both known and unknown viruses. In most of these methods, proteolysis is performed on intact viruses for varying incubation times, with protein molecular weights compared to those in standard databases. However, these techniques are both time-consuming and unreliable for viruses that show few biomarkers. Catherine Fenselau and colleagues at University of Maryland (College Park) recently proposed a new bioinformatics strategy to solve this problem (Anal. Chem. 2002, 74, 2529–2534). Their solution involves using a database of the calculated masses of peptides predicted from residue-specific protease cleavage (e.g., by trypsin) of all proteins in each microorganism of interest. In this strategy, the experimentally observed peptide masses are searched against the database to identify the virus directly. The researchers digested Sindbis virus AR339 (SIN) with a trypsin solution. At various digestion times, samples were subjected to MALDI-MS analysis. The thin-layer sample preparation technique was used for MALDI-TOF analysis, and the instrument was internally calibrated with known peaks corresponding to autolysis trypsin peptides. After digestion, the scientists compared their results with a database they constructed from the National Center for Biotechnology Information proteome databases of proteins from six different viruses: Lelystad virus, Sindbis virus (SIN) strain AR339, Ross River virus strain NB5092, human adenovirus type 5, tobacco mosaic virus 302

U2, and bacteriophage MS2. All experimentally observed peptide masses from the digested SIN solution were compared with the masses of the in silico-generated tryptic peptides from each virus within a range of 1000−4000 Da. Digestion of the SIN virus for only 2 min resulted in the observation of more than 20 tryptic peptides in a MALDI spectrum. Digestion of 5 minutes or longer did not increase the number of observed peptides. Two algorithms were tested for identification: a direct score-ranking algorithmone that links the highest number of matched peptide weights with the target organismand an algorithm that evaluates the probability of random matching. The SIN virus was

Cleave, separate, and search. A new strategy for virus identification involved partial proteolysis, mass spectrometry, and database queries. (Adapted from Anal. Chem. 2002,74, 2529–2534.)

unambiguously identified by either approach. The new method identifies whole virus samples in less than 5 minutes, using a MALDI-TOF MS with limited resolution. The scientists envision the construction of an on-line tryptic peptide database for all viruses with known genome sequences in the future, incorporating additional information such as protein copy number, post-translational modifica-

JournalofProteom e Research • Vol. 1, No. 4, 2002




tions, and virus topology.

M icrofabricated Cell Sorter Proteome complexity is often due to tissue heterogeneity, where each different cell type contributes its own protein milieu. Thus, the isolation of specific cells from a mixture would simplify proteomic analysis. Traditionally, this has been accomplished with techniques such as fluorescenceactivated cell sorters (FACS), but these systems suffer from the need for large samples, high background fluorescence, and the potential for cross-contamination. With this in mind, Stephen Quake and colleagues at the California Institute of Technology (Pasadena) developed an integrated microfabricated cell sorter using multilayer soft lithography (Anal. Chem. 2002, 74, 2451–2457). The cell sorter has two layers: a top piece that carries pneumatically controlled channels for the pumps and valves, and a bottom layer that carries the microfluidic lines (which form a T-shape) for sample injection, collection, and waste removal. The sorter is placed on an inverted microscope, and a laser is used as the excitation source. A photomultiplier tube detects the fluorescence of passing cells. As the cells move along through the sorter, they pass through the fluorescence detector at the junction of the

Celltrapping.A cell can be isolated within the detection junction by reversing the buffer flow at each detection event. (Adapted from Anal. Chem. 2002,74, 2451–2457.)

three sample lines. If no signal is detected, the pumps send the cell to the waste channel. If a cell fluoresces, however, the buffer flow is reversed, which sends the cell back through the detector. If fluorescence is detected a second time, the cell is then sent to the collection channel. The researchers tested their system on a mixed population of E. coli expressing either enhanced green fluorescent protein (EGFP) or p-nitrobenzyl (pNB) esterase. After sorting, the cells in the waste and collection channels were plated onto nutrient agar containing either ampicillin (upon which the EGFP cells would grow) or tetracycline (upon which the pNB cells would grow). Almost 500,000 cells could be sorted in a single run, recovery yields reached 50% for some experiments, and cells were enriched up to 89-fold. The size of the integrated cell sorter means that very small samples can be handled quickly and with reduced background fluorescence as compared to FACS. Furthermore, the detection optics offer superior sensitivity, and the simple fabrication and inexpensive materials make the unit disposable, eliminating problems of cross-contamination.