Labeled peptides

Acid hydrolysis cleaves the amide bonds of the 2 4 dimtrophenyl labeled peptide giving the 2 4 dimtrophenyl labeled N terminal ammo acid and a mixture of unlabeled ammo acids... 

Preparation of mixed peptide fibrils was similar for both of these labelled peptides 1% (by weight) was incubated at 37 °C with the other, unlabelled peptide in 10% CH3CN/H20atpH2.ForTTRio i9with l%dansyl-TTRio5 n5, a blue shift and dansyl anisotropy increase were observed, indicating the inclusion of dansyl-TTRios-i 15 into fibrils. CD spectroscopy proved that the stmcture was primarily p-sheet. 

Reaction of purified Ca " -ATPase with 0.3 mM NBD-Cl in the presence of 1 mM AMP-PNP and 1 mM CaCl2 caused inhibition of ATPase activity with the incorporation of 2= 15 nmol NBD-Cl per mg protein [335]. The inhibition was attributed to the binding of 7-8 nmol NBD-Cl/mg enzyme protein, corresponding to = 1 mol NBD-Cl per mol ATPase. The NBD-labeled enzyme was digested with pepsin and several NBD-labeled peptides were isolated [335]. All peptides contained the Gly-X (Cys) sequence that occurs only in one place in the Ca -ATPase, i.e., at Gly343-Cys344. Therefore NBD-Cl reacts with the same cysteine 344 residue that is also modified by maleimide derivatives [319]. The NBD modified enzyme had only 5-10% of the ATPase activity of the control ATPase, but the steady state concentration of the phosphoenzyme intermediate was only slightly reduced [335]. The Ca ... 

Figure 3.3. Structure of the ICAT reagent. The reagent contains a biotin affinity tag that is used to isolate ICAT-labeled peptides. The reagent also contains a linker that exists in a heavy (where X= deuterium) or light form (X= hydrogen) and a reactive group with specificity towards the thiol groups of cysteine residues. Figure adapted from Gygi et al. (1999).

Shimura, K., Kamiya, K., Matsumoto, H., Kasai, K. (2002). Fluorescence-labeled peptide pi markers for capillary isoelectric focusing. Anal. Chem. 74, 1046-1053. 

HPLC with microchip electrophoresis. Capillary RPLC was used as the first dimension, and chip CE as the second dimension to perform fast sample transfers and separations. A valve-free gating interface was devised simply by inserting the outlet end of LC column into the cross-channel on a specially designed chip. Laser-induced fluorescence was used for detecting the FITC-labeled peptides of a BSA digest. The capillary HPLC effluents were continuously delivered every 20 s to the chip for CE separation. 

Ohuchi Y, Katayama Y, Maeda M (2001) Fluorescence-based sensing of protein kinase A activity using the dual fluorescent-labeled peptide. Anal Sci 17 il465—i 1467... 

Farley RA, Tran CM, Carilli CT et al (1984) The amino acid sequence of a fluorescein-labeled peptide from the active site of (Na, K)-ATPase. J Biol Chem 259 9532-9535... 

In a similar fashion, steroids are molecules that have been investigated by disruption of FRET. The sensor is a double labeled peptide with cyclodextrin bound to one side chain. The latter keeps the fluorophores closely together by accommodating the coumarin into its cavity thereby ensuring efficient FRET. Steroids compete for the cavity of cyclodextrin and displace the coumarin reducing FRET efficiency. This model, although useful for in vitro applications, seems to be poorly selective for its application in biological samples [95],... 

Figure 16.4 A more advanced ICAT design uses an acid-cleavable spacer arm to facilitate elution of labeled peptides from a (strept)avidin affinity column. The use of 14C isotopes instead of deuterium labels permits precise reverse phase separations prior to mass spec that show no elution peak time differences between isotope-labeled and normal atom-labeled peptides.

Figure 16.5 A catch-and-release ICAT design incorporates a gem-methyl group and an isopropyl group on either side of a disulfide bond within its spacer arm. The hindered disulfide permits the use of standard reducing gel electrophoresis conditions using DTT without reduction. After purification on a (strept)avidin affinity column, however, the disulfide group can be cleaved with TCEP, which provides recovery of the labeled peptides prior to mass spec separation.

Figure 16.6 The solid phase ICAT reagent provides a thiol-reactive iodoacetyl group to capture cysteine peptides, a spacer containing stable isotopic labels, and a photo-cleavable group that can release the captured peptides for mass spec analysis. The VICAT mass tag is a solution phase labeling agent that also has a photo-cleavable site to release isolated peptides from a (strept)avidin affinity resin. This compound adds a fluorescent group to better detect labeled peptides as they are being isolated from a sample.

The use of PIR compounds to study protein interactions is a significant advance over the use of standard homobifunctional crosslinkers. The unique design of the PIR reagent facilitates deconvolution of putative protein interaction complexes through a simplified mass spec analysis. The software can ignore all irrelevant peak data and just focus analysis on the two labeled peptide peaks, which accompany the reporter signal of appropriate mass. This greatly simplifies the bioinformatics of data analysis and provides definitive conformation of protein-protein crosslinks. 

In a more general case, some signals from natural abundance 13C nuclei will dephase (5,na d) because of the finite probability that they are in close proximity of other 13C nuclei. Furthermore, some labeled peptide molecules are unfibrillized and we assume that their signals will not dephase (+Lu). Consequently, we can write... 

Frequency-selective REDOR (fsREDOR) is a very powerful technique developed for the study of 13C and 15N uniformly labeled peptides or proteins [92]. The basic idea of this technique is to combine REDOR and soft n pulses to recouple a selected 13C-15N dipole-dipole interaction in a multiple-spin system. Usually one could use Gaussian shaped pulses to achieve the required selective n inversions. Other band selective shaped pulses have been developed for a more uniform excitation profile [93]. In its original implementation, fsREDOR was used to extract the intemuclear distances of several model crystalline compounds [92], In the past few years, this technique has proven to be very useful for the study of amyloid fibrils as well. For the Ure2p10 39 fibril samples containing 13C and 15N uniformly... 

Abstract To understand how membrane-active peptides (MAPs) function in vivo, it is essential to obtain structural information about them in their membrane-bound state. Most biophysical approaches rely on the use of bilayers prepared from synthetic phospholipids, i.e. artificial model membranes. A particularly successful structural method is solid-state NMR, which makes use of macroscopically oriented lipid bilayers to study selectively isotope-labelled peptides. Native biomembranes, however, have a far more complex lipid composition and a significant non-lipidic content (protein and carbohydrate). Model membranes, therefore, are not really adequate to address questions concerning for example the selectivity of these membranolytic peptides against prokaryotic vs eukaryotic cells, their varying activities against different bacterial strains, or other related biological issues. 

Fig. 1 Solid-state NMR structure analysis relies on the 19F-labelled peptides being uniformly embedded in a macroscopically oriented membrane sample, (a) The angle (0) of the 19F-labelled group (e.g. a CF3-moiety) on the peptide backbone (shown here as a cylinder) relative to the static magnetic field is directly reflected in the NMR parameter measured (e.g. DD, see Fig. 2c). (b) The value of the experimental NMR parameter varies along the peptide sequence with a periodicity that is characteristic for distinct peptide conformations, (c) From such wave plot the alignment of the peptide with respect to the lipid bilayer normal (n) can then be evaluated in terms of its tilt angle (x) and azimuthal rotation (p). Whole-body wobbling can be described by an order parameter, S rtlo. (d) The combined data from several individual 19F-labelled peptide analogues thus yields a 3D structural model of the peptide and how it is oriented in the lipid bilayer...

Fig. 2 Mechanically oriented bilayer samples as a membrane model for ssNMR. (a) Illustration of the hydrated lipid bilayers with MAPs embedded, the glass supports, and the insulating wrapping, (b) A real sample consists of 15 stacked glass slides, (c) Schematic solid-state 19F-NMR lineshapes from an oriented CF3-labelled peptide (red), and the corresponding powder lineshape from a non-oriented sample (grey), (d) Illustration of typical orientational defects in real samples - the sources of powder contribution in the spectra...

FIGURE 6.51 Determination of Vmax and Km for ATP. A Michaelis-Menten plot of PKA phosphorylation of labeled peptide substrate was used to determine Vmax and Km in the presence and absence of three concentrations of inhibitor H-89. 

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