Peptides, structure-directing

NMR and kinetic studies have been conducted with the hope of providing more details about the position and conformation of the polypeptide substrate in cAMP-dependent protein kinase. These have served to narrow down the possible spatial relationships between enzyme bound ATP and the phosphorylated serine. Thus, a picture of the active site that is consistent with the available data can be drawn (12,13,66,67). Although these studies have been largely successful at eliminating some classes of secondary polypeptide structure such as oi-hellces, 6-sheets or an obligatory 6-turn conformation 66), the precise conformation of the substrate is still not known. The data are consistent with a preference for certain 6-turn structures directly Involving the phosphorylated serine residue. However, they are also consistent with a preference or requirement for either a coil structure or some nonspecific type of secondary structure. Models of the ternary active-site complexes based on both the coil and the, turn conformations of one alternate peptide substrate have" been constructed (12). These two models are consistent with the available kinetic and NMR data. 

Iodination of PIR (147) showed 1 residue buried, Tyr 25, and all others iodinated at least to the monoiodotyrosyl form. Pepsin-inactivated RNase also has only one abnormal tyrosyl by titration which is thus assumed to be 25. Iodination of RNase-S is very similar to RNase-A in the early stages (lift). Extensive iodination leads to dissociation of the protein and peptide components. Direct iodination of S-protein indicated that all 6 tyrosyl residues were accessible, in this sense comparable to urea-denatured RNase-A. Substantial structural changes must be involved for both S-protein and PIR if Tyr 97, in particular, is to become susceptible to attack (see Section IV,B,3). 

The above results illustrate the diversity of unnatural tricks that chemists have used recently in order to help peptidic molecules adopt a well defined structure in solution. Efforts were directed to the development of both single and multiple peptide structures. 

Resonance Raman Spectroscopy. If the excitation wavelength is chosen to correspond to an absorption maximum of the species being studied, a 102—104 enhancement of the Raman scatter of the chromophore is observed. This effect is called resonance enhancement or resonance Raman (RR) spectroscopy. There are several mechanisms to explain this phenomenon, the most common of which is Franck-Condon enhancement. In this case, a band intensity is enhanced if some component of the vibrational motion is along one of the directions in which the molecule expands in the electronic excited state. The intensity is roughly proportional to the distortion of the molecule along this axis. RR spectroscopy has been an important biochemical tool, and it may have industrial uses in some areas of pigment chemistry. Two biological applications include the determination of helix transitions of deoxyribonucleic acid (DNA) (18), and the elucidation of several peptide structures (19). A review of topics in this area has been published (20). 

Thus, the question in coiled-coil prediction and design is what specific replacements are superimposed on the basic HPPHPPP pattern to direct the functional oligomerization state This question was first tackled by Conway and Parry, who analyzed natural coiled-coil sequences that formed dimers and trimers (Conway and Parry, 1990, 1991). Woolfson and Alber (1995) advanced this approach by comparing amino-acid profiles for these two structures directly. The work that made the biggest impact on this issue, however, was the collaborative experimental study from the Kim and Alber laboratories using the GCN4 leucine-zipper peptide model system and mutants thereof. 

Note that, by convention, in writing peptide structures, each peptide bond is written in the following direction ... 

D. Catalytic, Structure-Directing Peptides Confirm and Extend these... 

This approach offers a number of significant advantages. Since whole cells are used as the affinity support, the receptors are likely to be in their native conformation, allowing the isolation of peptide ligands directed against conformationally structured epitopes [70, 71]. Moreover, the technique leads to the identification of cell surface markers which distinguish otherwise similar cell types such as antigens, epitopes, or receptors, without the need to either identify or purify a particular receptor in advance [38, 42, 43, 72]. 

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. 

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... 

Other proteases employ the same catalytic strategy. Some of these proteases, such as trypsin and elastase, are homologs of chymotrypsin. In other proteases, such as subtilisin, a very similar catalytic triad has arisen by convergent evolution. Active-site structures that differ from the catalytic triad are present in a number of other classes of proteases. These classes employ a range of catalytic strategies but, in each case, a nucleophile is generated that is sufficiently powerful to attack the peptide carbonyl group. In some enzymes, the nucleophile is derived from a side chain whereas, in others, an activated water molecule attacks the peptide carbonyl directly. 

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