currents
with five major peaks with varying intensitiesone for the Despite the importance of metal ions in protein chemistry, very unbound protein and four representing the possible calciumfew methods allow direct determination of protein−metal ion bound molecules (1–4 Ca2+ ions)indicating different binding interactions or exact stoichiometric binding ratios of the metaffinities for each site. als. MS, particularly ESI-MS, is an effective tool for studying The RILED technique offers the same advantage as SEC in noncovalent interactions like protein−metal binding; however, terms of on-line removal of involatile salts prior to ESI but does sample preparation, which requires desalting and buffer exchange not have SEC’s ability to separate other potential protein soluof the protein solution before ESI, can be time- and labor-intention components. However, when the mixture is not complex sive. Scientists from the Mayo Clinic (e.g., calbindin D28K), SEC separations are not necessary. In such cases, the (Rochester, MN) recently demonrelative quickness of RILED (with typstrated the use of two techniquesonical elution times of 2−5 min comline size exclusion chromatography pared with SEC’s 15−60 min) makes (SEC) and rapid in-line desalting it a worthwhile alternative. In this (RILED)coupled to µESI for a more study, the Mayo researchers were able simplified, one-step analysis of proto use RILED-µESI-MS and produce tein−metal interactions (Rapid Comm. results very similar to those yielded by Mass Spectrom. 2003, 17, 267–271). SEC for calbindin D28K calcium interThe Mayo team focused mostly on actions, but in a shorter time. And for calbindin D28K, which is a high-affinity calcium-binding protein that coneven more direct and rapid analysis, tains six potential metal-binding sites. the team bypassed the incubation step They incubated the protein with caland simply added different concencium and directly analyzed it with trations of Ca2+ to a RILED mobile phase with protein and EDTA. The SEC/µESI-MS. The resulting spectrum ensuing spectra demonstrated the had a dominant ion peak equaling the presence of unbound protein as well mass of calbindin D28K plus four Ca2+ ions, which supports earlier stoichioas successive Ca2+ additions to the metric findings for this system. molecule as the ion concentration in To determine whether SEC/µESIthe mobile phase was increased. MS could give any sense of the relative Once the mobile phase conditions binding affinities of the protein sites, Sizing up conditions.The SEC/µESI-MS of 15 µM calbindin are optimized, say the authors, sysEDTA was added to the chromato- D28K in SEC buffer containing (A) 150 µM Ca2+ and (B) 150 tematic titration studies can be cargraphic mobile phase to act as a com- µM Ca2+ plus 1 µM EDTA. (Reproduced with permission from ried out to quickly obtain valuable petitive calcium binder. The addition Benson, L. M.; et al. Rapid Comm. Mass Spectrom. 2003, 17, information about metal interactions of one µM EDTA produced a spectrum 267–271. Copyright 2003 John Wiley & Sons, Ltd.) with a range of different proteins.
Protein−M etalInteractions
On-Chip,Unlabeled Laser-induced fluorescence is capable of extremely low detection limits for separations accomplished with chip-based CGE. However, chemical derivatization steps are generally required to incorporate fluorescent moieties. This not only adds timeconsuming chemistry to the process, but also might complicate the separation analysis when multiple reaction sites in proteins result in multiple labeled derivatives with different electrophoretic mobilities. Thus, a universal chipbased detector is sought that © 2003 American Chemical Society
is comparably sensitive but works with label-free proteins. Darryl Bornhop and colleagues from Texas Tech University (Lubbock) have reported such a device using backscatter interferometry for on-chip SDS-CGE analysis based on refractive index (RI) detection (Electrophoresis 2003, 24, 865–873). Their onchip interferometric backscatter detector (OCIBD) exhibits attomole sensitivity for unlabeled proteins and polypeptides. The OCIBD system works on the basis of the interaction of a He–Ne laser beam with
two intersecting microfluidic channels photolithographically etched in a fused-silica plate. The backscattered light from the channel takes the form of a high-contrast interference pattern that confers information related to the bulk properties of the analytecontaining fluid. Using egg albumin as an analyte, the researchers experimented with two SDS-CGE sample loading techniques: gated valve injection, which involves sample flowing perpendicular from reservoir 1 to 2, and pinched injection, which entails forcing the sam-
ple to migrate across the channel intersection between reservoirs 2 and 3. The gated valve approach demonstrated an 11-amol detection limit and the pinched injection method showed a 17-amol limit, both highly sensitive and comparable with previous results obtained with fluorescence. Using the pinched method, the researchers subsequently injected a mixture of five unlabeled proteins— calmodulin, α-lactalbumin, pepsinogen, egg albumin, and BSA—into the SDS-CGE system. The proteins, ranging from 9 to 66 kDa, were well
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currents resolved with sharp RI signals within 100 s. Detection limits ranged from 0.95 pg (110 amol) for calmodulin to 7.0 pg (100 amol) for BSA. These experiments were performed without any active temperature control, which, the researchers say, would further increase the performance of the system, because Joule heat produced by applying voltage to the CE channels can perturb the RI detector. Even without this refinement, however, they assert that the OCIBD measures up to existing methods for the on-chip analysis of proteins and will be of particular use when solute derivatization is not a viable option.
M ore Than the Sum Using a method called native chemical ligation (NCL), researchers can join two peptidic or nonpeptidic compounds, with the simple requirement that one compound carry a thioester moiety and the other an aminothiol group. The compounds couple when the aminothiol nucleophile attacks the thioester, resulting in the loss of a thioalkyl group. An intramolecular acyl shift then results in an irreversible amide bond and a cysteine at the junction. Because this reaction occurs under aqueous conditions and without protecting groups, it has become a popular replacement for the more standard protected block synthesis. The need for a cysteine at the N-terminus of one of the reactants, however, limits the scope of the method. To address this problem, Jason Betley and colleagues at Adprotech (Little Chesterford, U.K.) developed a linker molecule that carries a pseudo-Nterminal cysteine at the peptide C-terminus (J. Pept. Sci. 2003, 9, 221–228). The researchers reacted an Fmoc-Lys(Boc)-OH that
Double yourpleasure.The native chemical ligation of a thioester-containing peptide with a small-molecule linker results in a pseudosymmetrical dimer. (Adapted with permission from J. Pept. Sci. 2003,9, 221–228. Copyright 2003 John Wiley & Sons, Ltd.)
was deprotected at the εnitrogen with an activated ester of Boc-Cys(Trt)-OH to generate a pseudo-cysteine monomer that could be incorporated into standard peptide synthesis protocols. They then incorporated the monomer into a short peptide and performed NCL on a thioester of enhanced green fluorescent protein, using SDS-PAGE to show that the protein was indeed larger by approximately the size of the peptide (~2 kDa) after ligation. To prove that this ligation occurred by NCL, the researchers showed that the larger protein complex was impervious to reduction by mercaptoethanol. The researchers then synthesized a small-molecule linker that could perform NCL at both ends via a true N-terminal cysteine and a C-terminal pseudo-cysteine monomer, thus allowing them to form a peptide homodimer by reacting a molar excess of a single thioester. The researchers could also form a heterodimer by first reacting their linker with one peptide, purifying the product and then reacting it with a second peptide or protein. According to the researchers, “There is no theoretical limit to the number of
reactive NCL components installed in a peptide sequence, leaving the way open to use this chemistry to generate a chemical scaffold for the attachment of multiple other molecules in a stable native fashion.”
CrystalScreens
To address this problem, Eila S. Cedergren-Zeppezauer and colleagues at Lund University (Sweden) developed a method for rapidly screening crystallization parameters— combinations of temperatures, solvent compositions, and degrees of supersaturation—for a particular protein, using a minimal amount of protein material (Anal. Chem. 2003, 75, 1733–1740). They also developed a flow-through microdispenser to introduce solutions of crystallizing agents into a droplet of protein solution. A piezoelectric element activates a piston, which pushes crystallizing agent solution through a silicon microchannel, ejecting 50- to 100-pL droplets from a nozzle at a rate of up to several kHz (65 pL and 50 Hz were used for the supersaturation tests). A drop of liquid containing the test protein, typically 0.25–1.5 µL, was levitated in an ultrasonic standing wave so that the drop was surrounded on all sides by the ambient atmosphere. While a microscope equipped with a CCD camera monitored the process, the researchers sprayed the protein drop with the crystallizing agent and tracked the onset of supersaturation using right-angle light-scattering (RALS) measurements. Because the pro-
Single-crystal X-ray diffraction analysis is still the gold standard for protein structure determination, even though growing suitable crystals is often problematic. Protein molecules, with their flexibility and complex shapes, can be difficult to crystallize, and the mechanisms by which they precipitate from solution and form crystals are poorly understood. Protein crystallization is often conducted using a trial-and-error approach, and those proteins that do not crystallize using known recipes are Clouding the issue.Researchers used light scattering to abandoned in determine under what conditions a protein will precipifavor of more tate from solution (a: clear protein solution; b–e: increastenable poly- ing precipitation; f: maximum precipitation). (Adapted with permission from Anal. Chem. 2003,75, 1733–1740.) peptides. JournalofProteom e Research •Vol. 2, No. 3, 2003
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currents tein solution achieves supersaturation so rapidly, crystalline nuclei do not have time to form, and only amorphous precipitation occurs. This gives a quick indication of the precipitation limit of the protein under given conditions and generates a rough estimate of the boundaries between low and high supersaturation states, screening for combinations that can be studied in more detail during subsequent nucleation and optimization experiments. The system was tested on four enzymes. Poly(ethylene glycol), various organic and inorganic salts, and 2-methyl2,4-pentanediol were used as crystallizing agents. RALS intensity was plotted versus time to track the onset of precipitation. After screening various combinations of solutions using the levitated-drop procedure, the researchers selected the best candidates and tested them in tissue culture plates for nucleation using batch crystallization under paraffin oil.
Antibody M im ics
to nonimmunoglobulin scaffolds that are potentially easier to produce and intrinsically more stable. Pär Nordlund and colleagues at Stockholm University and the Royal Institute of Technology (Stockholm) used phage display technology to create a library of proteins (affibodies) that will bind to a variety of protein ligands. Specifically, they randomized 13 residues on one side of protein Z, a 58-residue domain from staphylococcal protein A (SPA); using SPA as a selection target, they generated a protein A-specific affibody. To understand how affibodies work, the researchers determined the structure of an affibody-protein Z complex by X-ray crystallography (Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3191–3196). The researchers found that helices 1 and 2 of the affibody packed tightly against helix 1 and much of helix 2 of protein Z, forming a distorted four-helix bundle. Not surprisingly, the surfaces of the two interfaces were largely populated with hydrophobic residues. Also, the interaction surfaces are very complementary in shape, with bulky residues on one face fitting into deep cavities on the other. The researchers then compared their structure with a crystal structure of a protein
The ability of the immune system to generate a vast array of antibodies specific for an equal plethora of ligands has led to the use of antibodies as a critical tool in research. This is nowhere better exemplified than with antibody-based protein chips. In the past few years, in vitro antibody production has become the norm, but problems with immunoglobulin folding and stability have limited the antibody-based repertoire. For Inagroove.A stereo image of the ZSPA affibody helices this reason, 1 and 2 over the surface of protein Z. (Adapted with perresearchers have mission from Högbom, M.; et al. Proc. Natl. Acad. Sci. started to shift U.S.A. 2003, 100, 3191–3196. Copyright 2003 National Academy of Sciences, U.S.A.) their attentions 238 JournalofProteom e Research •Vol. 2, No. 3, 2003
Z–IgG complex and found that although the binding surfaces of the IgG and affibody have completely different folds (loops and β-strands vs α-helical), the two proteins bind to the same part of protein Z. The researchers explain that this effect might be caused in part by a selection bias because they used eluted protein Z-binding affibodies with human monoclonal IgG. However, they also suggest that this might be further evidence for the existence of phage display-binding hot spots, which have been seen in studies by other groups. The researchers believe that because of the binding specificity and ease of generation and production, “in vitro evolved binding proteins should constitute a valuable tool and provide possibilities for novel applications in biotechnology.”
M ethylation and Apoptosis High mobility group A (HMGA) nuclear proteins are very abundant in cancer cells but are barely detectable in normal differentiated cell lines, making them good diagnostic tools and possible therapeutic targets. The HMGA family consists of three ~11-kDa DNA-binding proteins that are encoded by two genes. The DNA-binding region of these proteins consists of three highly cationic regions known as AT-hooks, which bind to AT-rich regions of DNA. In early studies to investigate the function of these proteins, Riccardo Sigarra and colleagues at the Università di Trieste and the Università di Undine (Italy) found that protein hyperphosphorylation during apoptosis caused the HMGAs to detach from the DNA. To expand on this analysis, the same researchers recently exposed four tumor cell lines to various apoptotic
agents and isolated HMGA1a protein from these cells (Biochemistry 2003, 42, 3575–3585). They then treated the protein with alkaline phosphatase to remove the phosphate groups, digested the treated HMGA1a protein with trypsin, and analyzed the resulting peptides with HPLC-MS. The researchers found that a specific methylation event occurs at arginine-25— located in the first AT-hook of the protein—and that this event occurred more often (up to 2-fold) in apoptotic cell lines than in the control lines. This was in sharp contrast to the other HMGA proteins, none of which were methylated. The researchers then added recombinant HMGA1a protein to extracts of cells treated with tritiated S-adenosylmethionine and one of the apoptotic agents and again found methylation at arginine-25. This result indicated the presence of an active methyl transferase in the induced cells. In addition, the researchers discovered that HMGA1a methylation during apoptosis appeared to be coupled to the dephosphorylation of the protein in each of the cancer cell lines that they monitored. Of the other HMGA proteins that they examined, only the closely related HMGA1b showed similar phosphorylation patterns. Performing methylation inhibition experiments using adenosine dialdehyde, the researchers showed that methylation is not one of the causal events in apoptosis but is likely related to the cell death-induced transformation of chromatin architecture. Although no obvious function for HMGA1a methylation was presented, the authors postulate that it “could be an additional structural contribution to the so-called histone code that regulates the genereading machinery.”
