Secondary Structures, Protein 3-10 Stretch

Upon biosynthesis, a polypeptide folds into its native conformation, which is structurally stable and functionally active. The conformation adopted ultimately depends upon the polypeptide s amino acid sequence, explaining why different polypeptide types have different characteristic conformations. We have previously noted that stretches of secondary structure are stabilized by short-range interactions between adjacent amino acid residues. Tertiary structure, on the other hand, is stabilized by interactions between amino acid residues that may be far apart from each other in terms of amino acid sequence, but which are brought into close proximity by protein folding. The major stabilizing forces of a polypeptide s overall conformation are ... 

Large portions of most protein structures can be described as stretches of secondary structure (helices or /3 strands) joined by turns, which provide direction change and offset between sequence-adjacent pieces of secondary structure. Tight turns work well as a-a and a-fi joints, but their neatest application is at a hairpin connection... 

Secondary structures are formed by short stretches of residues. These substructures make up sequentially proximal components of proteins, and they have shapes. There are various forms of protein secondary structures, e.g., helices (the most common of which is the a-helix), P-sheets, p-tums, Q-loops, and some that remain unclassifiable and are typically referred to as random coil or loop regions. A complex combination of attractive and repulsive forces between close and more distant parts of the structure affects the resultant shape of secondary structures, and predicting secondary structure from knowledge of the linear amino acid sequence alone remains a tremendous challenge. 

Vibrational spectroscopy has been used in the past as an indicator of protein structural motifs. Most of the work utilized IR spectroscopy (see, for example, Refs. 118-128), but Raman spectroscopy has also been demonstrated to be extremely useful (129,130). Amide modes are vibrational eigenmodes localized on the peptide backbone, whose frequencies and intensities are related to the structure of the protein. The protein secondary structures must be the main factors determining the force fields and hence the spectra of the amide bands. In particular the amide I band (1600-1700 cm-1), which mainly involves the C=0-stretching motion of the peptide backbone, is ideal for infrared spectroscopy since it has an large transition dipole moment and is spectrally isolated... 

The amide I mode is most widely used in studies of protein secondary structure [10, 108]. This mode gives rise to infrared band(s) in the region between 1600 cm-1 to 1750 cm 1 and is predominantly due to the CO stretching mode. The major factor responsible for conformational sensitivity of the amide I bond is coupling between... 

The mid-lR region (1800-800cm ) -also referred to as the fingerprint region-contains the stretching vibration of the ester linkage of lipids at around 1740 cm , and the most dominant spectral feature, the amide 1 protein vibration centered around 1655 cm . This vibration is the exciton-coupled C=0 stretching manifold, and consequently, is sensitive for both peptide and protein secondary structure. 

Implicit in the kinetic mechanism proposed here is the idea that a helix (probably an a-helix) is the main protein secondary structure and that all the other secondary structures arise from the breaking and destabilization of the a-helices. This new kinetic mechanism may explain the high helidty of short polypeptides that are part of jS-strands, as well as the presence of a-helical intermediates in the folding of predominantly j8-sheet proteins. The hierarchical nature of protein structure is also a natural consequence of the mechanism since that main secondary structure is present from the beginning and the tertiary structure results from the breaks and, either the packing of the helical stretches that arise in that early process, or the packing of the jS-sheets that form later when the first process happens to lead to unfavourable side chain interactions. 

It is thought that portions of the chain, such as small stretches of secondary structure, might serve as nucleation sites around which the rest of the protein will fold so that folding may be cooperative. 

The amide I band was chosen for detailed analysis as its position is sensitive to protein secondary structure. The band arises predominately from v(C=0) stretching of the carbonyl group within the peptide (CONH) bond. Other minor contributing factors arise from v(CN) out-of-plane, 5(CCN) and 8(NH) in-plane vibrations (1). The broad nature of the amide I band is attributed to the presence of a number of secondary structures within the sample. Derivatives were used to deconvolve spectral band widths and positions. This resolution technique, together with deconvolution and curve fitting, is particularly useful for resolving components within a broad band envelope. 

The keratins are a specialized group of structural fibrous proteins that are characterized by their high cystine content and can be classified according to the amount of sulfur present in the protein. Structures such as wool, hair, hooves, horns, claws, beaks and feathers are classified as hard keratins because the sulfur concentration in these proteins is greater than 3%. Keratin proteins containing less than 3% sulfur, such as the stratum corneum (the outermost layer of skin), are classified as soft keratins. Although these keratin proteins have a similar molecular composition major spectral differences have been observed in the intensities of the C-S and S-S stretching vibrations the conformation of the disulfide bond and the position, line shape and bandwidth of vibrations associated with the proteins secondary structure. 

In addition, for solid samples or peptides in nonaqueous solvents, the amide II (primarily in-plane NH deformation mixed with C—N stretch, -1500-1530 cm-1) and the amide A (NH stretch, -3300 cm-1 but quite broad) bands are also useful added diagnostics of secondary structure 5,15-17 Due to their relatively broader profiles and complicated by their somewhat weaker intensities, the frequency shifts of these two bands with change in secondary structure are less dramatic than for the amide I yet for oriented samples their polarization properties remain useful 18 Additionally, the amide A and amide II bands are highly sensitive to deuteration effects. Thus, they can be diagnostic of the degree of exchange for a peptide and consequently act as a measure of protected or buried residues as compared to those fully exposed to solvent 9,19,20 Amide A measurements are not useful in aqueous solution due to overlap with very intense water transitions, but amide II measurements can usefully be measured under such conditions 5,19,20 The amide III (opposite-phase NH deformation plus C—N stretch combination) is very weak in the IR and is mixed with other local modes, but has nonetheless been the focus of a few protein-based studies 5,21-26 Finally, other amide modes (IV-VII) have been identified at lower frequencies, but have been the subject of relatively few studies in peptides 5-8,18,27,28 ... 

Many experiments have been carried out by using this setup the stretching of single DNA molecules, the unfolding of RNA molecules or proteins, and the translocation of molecular motors (Fig. 2). Here we focus our attention on force experiments where mechanical work can be exerted on the molecule and nonequilibrium fluctuations are measured. The most successful studies along this line of research are the stretching of small domain molecules such as RNA [83] or protein motifs [84]. Small RNA domains consist of a few tens of nucleotides folded into a secondary structure that is further stabilized by tertiary interactions. Because an RNA molecule is too small to be manipulated with micron-sized beads, it has to be inserted between molecular handles. These act as polymer spacers that avoid nonspecific interactions between the bead and the molecule as well as the contact between the two beads. 

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