Analytical Currents: Protein structures by ATR FT-IR

easy access to the internal reflection element (IRE). The holder can be configured for thin-film ATR or as a thermostated 125-uLflow cell for the solu...
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(A) Schematic of a microfluidic network for the patterned delivery yf reactants. (B) A ΜFN on a silicon substrate.

A motif for metals Metal sensors for use in biological or environmental samples must be selec­ tive enough to measure the desired metal, even in the presence of compet­ ing cations. Barbara Imperiali and co­ workers at the California Institute of Technology used the tripeptide se­ quence glycine-glycine-histidine to mimic the copper-binding properties of the amino terminal Cu(II)- and Ni(II)binding (ATCUN) motif of the serum albumins to develop chemosensors. Because the ATCUN motif selec­ tively binds Ni(II) and Cu(II), intramo­ lecular quenching of an incorporated fluorophore can signal metal binding. The Caltech researchers incorporated a 5-(dimethylamino) naphthalene-1sulfonamide fluorophore into a family of pentapeptides based on the ATCUN motif. In each of the pep­ tides, the N-terminal amino acid was replaced with a homologous series of residues that contain a side chain amine for fluo­ rophore attachment The fluorescence decreased linearly until the chemosensor was saturated at which noint no further decrease was observed

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the surface. Following the surface derivatization, the polymer was quickly peeled off. Bovine serum albumin was used to block potential sites of adsorption outside the pattern of the uFN and prevent nonspe­ cific adsorption of secondary antibodies. Fluorescence measurements of tagged antigen-antibody pairs indicated that sur­ face derivatization occurred only near the opening of the conduit, although the solu­ tion had filled the entire capillary. Varia­ tions in the conditions suggested that the absence of a continuous flow—rather than electrostatic repulsion between the walls of the uFN and charged protein—was the culprit. By including a "flow-promoting pad" connected to the end of the array of channels they succeeded in derivatizing the entire length of the conduit. A key fea­ ture of the uFN constructed with PDMS is that the quality of the seal allows the pat­ terning of a wide variety of surfaces (I. Am Chem Soc 1 9 9 8 120 500-08)

The metal binding could be reversed by adding excess EDTA. Altering the peptide backbone resulted in a chemosensor with a linear response to Cu(II) over the range 100-1000 nM. Shorter linkers from the fluorophore to the peptide result in more efficient quenching, with a larger change in fluorescence for Cu(II) than for Ni(II) binding. Moreover, the divalent cations of several other metals have no effect on the chemosensor fluorescence. The researchers prepared immobi­ lized analogues on solid surfaces, which permit chemosensor regeneration with simple washing procedures. The solidphase method will allow rapid screening of chemosensors by combinatorial methods. (J. Am. Chem. Socc.998, 120, 609-10)

Typical quenching assay for a chemosensor immobilized on PEGA-1900 resin.

Analytical Chemistry News & Features, April 1, 1998

Protein structures by ATR FT-IR Transmission FT-IR has become a popular method for determining the secondary structure of proteins based on amide I analyses, but it presents a difficult choice. If H 2 0 is used as the solvent, then high pro­ tein concentrations are required be­ cause of an intense band around 1640 cm"1, whereas D 2 0 as a solvent offers different H-bonding properties than does H.,0, is hygroscopic, and requires that the amide protons in the protein be completely exchanged for deuterons Attenuated total re­ flectance (ATR) FT-IR on the other hand offers the advantages of trans­ mission FT-IR plus it can provide data with low concentrations of proteins in the presence of strongiv absorbine" solutes and with nontranssamples Keith Oberg and Anthony Fink of the University of California at Santa Cruz describe experimental proce­ dures in which this approach is used for proteins in solution and evaluate die accuracy of the method by collect­ ing spectra from 13 proteins. Spectra were collected with a horizontal outof-compartment holder, which allows easy access to the internal reflection element (IRE). The holder can be configured for thin-film ATR or as a thermostated 125-uLflow cell for the solution work. In the solution approach, pro­ teins or peptides adsorb to the sur­ face of the IRE. A working model of protein-IRE interaction is pre­ sented and then used to develop a method for extracting structural information from solution ATR spectra, which are collected with solutions containing < 0.3 mg/mL of protein. Using a partial leastsquares analysis, the researchers report relative mean and standard deviation errors for the basis set structural analysis (e.g 6.3% for α-helix and 5 9% for p-sheet/extended structure) that are similar to values from comparable analyses with transmission FT-IR (Anal Biochem 1 9 9 8 , 256, 92—106)