Peptide structure investigations

Within five years of the verification of VCD in solutions, first attempts were made to apply the technique to study biomolecular solution conformation in aqueous solution. The first samples studied as solutions in D.O were simple amino acids and di-and tripeptides [26]. However, due to instrumental limitations, VCD was collected only in the C-H and N-H stretching region (2.5 to 3.5 fim). Although these early studies were necessary for the development of the experimental technique, it was not until experimental advances allowed the detection of VCD in the 6 fxm region that the full potential of VCD was realized for peptide structural investigations. 

Conformation and stability of thiopeptides (formed by replacing the amide oxygen atom with a sp2 sulfur atom) have been investigated by calculations. It has been confirmed that insertion of a thioamide linkage into a peptide structure is not conformationally neutral. It is predicted to be more rigid than peptide and produces substantial changes in peptide structure, primarily in the residues on the C-terminal side of the thioamide.78 80... 

The major strategies to enhance transmucosal peptide and other drug absorption include (a) coadministration with protease inhibitors, (b) the use of membrane permeation enhancers, (c) coadministration with a combination of absorption enhancers and protease inhibitors, (d) modification of peptide structure to improve metabolic stability or membrane permeation, and (e) use of nano- or microparticles [27], Some of these strategies have been investigated using the in situ rat model. 

While functional (immunological) mimicry has been established, the basis of mimicry on the molecular level remains to be explained. Several hypotheses have been put forward one of the earliest was that the side chains of aromatic amino acid residues might mimic the hydrophobic faces of the pyranosyl rings of carbohydrates. Before 1997, no structural evidence was available to support or discount these hypotheses. The nature of peptide-carbohydrate mimicry on the molecular level became the subject of structural investigations, and the resulting studies along with functional data will be discussed below. 

Subsequent to substrate binding, a promoted-water mechanism is favored for the hydrolysis of the scissile peptide linkage based on the results of chemical, kinetic, and structural investigations of carboxypeptidase A. A general mechanism is shown in Fig. 31, in which the zinc-bound water of the native enzyme is a nucleophile promoted both by zinc and by the general base Glu-270 (Christianson and Lipscomb, 1989). 

While opioid peptides have been very useful for investigating the pharmacology of different opioid receptor subtypes, pharmacological investigations have established that no pharmacodynamic advantage is to be expected from opioid peptides with respect to analgesic activity or side-effects. Furthermore, they have their own shortcomings with respect to potential clinical applications. Most importantly their peptidic structure usually prohibits administration by the oral or transdermal route, which are the routes of choice for pain treatment. 

De Jong, R., Rijkers, D. T. S., and Liskamp, R. M. J. (2002) Automated solid-phase synthesis and structural investigation of /3-peptidosulfonamides and /3-peptidosulfonamide//J-peptide hybrids /J-peptidosulfonamide and /3-peptide foldamers are two of a different kind. Helv. Chim. Acta 85,4230 1243. 

Once it was established that a number of enzymes contained non-peptidic structures, and that these features were present as binding sites for catalytic metals, researchers started to look for simple analogues that could be used as models for the more complex natural systems. The motivation behind these investigations was to gain a greater understanding into how the natural systems work by reducing the active... 

Considerable effort has been directed towards the stabilization of therapeutic peptides and proteins both in vitro and in vivo. Several methods of modifying peptide structure to improve metabolic stability have been investigated, as outlined in Section 1.6.1. 

Figure 15 Structures of the three peptide samples investigated by two-dimensional IR spectroscopy a de novo cyclic penta peptide (cycto-Mamb-Abu-Arg-Gly-Asp), apamin, and scyllatoxin.

We have investigated peptides whose structures were known beforehand from NMR or x-ray spectroscopy and related these structures to 2D-IR spectroscopy. Ultimately, one would like to deduce the structure of an unknown sample from a 2D-IR spectrum. In the case of 2D NMR spectroscopy, two different phenomena are actually needed to determine peptide structures. Essentially, correlation spectroscopy (COSY) is utilized in a first step to assign protons that are adjacent in the chemical structure of the peptide so that J coupling gives rise to cross peaks in these 2D spectra. However, this through-bond effect cannot be directly related to the three-dimensional structure of the sample, since that would require quantum chemistry calculations, which presently cannot be performed with sufficient accuracy. The nuclear Overhauser effect (NOE), which is an incoherent population transfer process and has a simple distance dependence, is used as an additional piece of information in order to measure the distance in... 

Protein crystal structures are archived in the Protein Data Bank (PDB) (Bernstein et al. 1977 Berman et al. 2000). About 5 per cent of the approximately 14 000 (December 2000) entries ( 12 500 proteins, peptides, and viruses, 900 nucleic acids, 600 pro-tein/nucleic acid complexes, 20 carbohydrates) contain the qualifier form in the compound name/descriptor field, and most of those refer to polymorphic varieties. In biomolecular crystallography great efforts are expended varying crystallization conditions in the attempts to obtain single crystals suitable for structural investigations... 

To understand the function of membrane-active peptides, it is important to know the structure and orientation of the peptide in the membrane. As is evident from Figure 18.1, it is possible to distinguish between, for example, carpet and pore mechanisms of action by determining the peptide s orientation in the membrane. Various techniques, such as electron spin resonance (ESR) [35], infrared (IR) spectroscopy [36-38], circular dichroism (CD) [35, 39,40], and solid-state NMR (SSNMR) [4-7] are used to investigate membrane-active peptides in a quasi-native lipid bilayer environment. In the following sections, methods to determine peptide structure and orientation are presented. 

The present review concentrates on the uses of nitrogen NMR studies in the structural investigations of peptides and similar compounds. Many of the molecules of interest are insufficiently soluble to permit solution-state NMR studies and thus solid-state measurements are the method of necessity. An... 

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