Conformational studies of the secondary structure

It is possible to perform more different analysis for the characterization of the secondary structure.

The most important are the NMR, IR, circular dichroism and X-ray.

1.6.1 MR studies

Since the hydrogen bonds are the principal responsible for the formation of the secondary structure, signals of amide protons are fundamental for the understanding of the peptide organization.

1.6.2 Choice of the solvent

The choice of the solvent is of fundamental importance in the NMR studies. In effect, the solvent can largely affect the ability of a peptide to form a secondary structure, and it is also quite commune that a peptide shows different conformations in different solvents.

1.6.3 2D MR

2D NMR analysis is one of the most powerful tools for the study of the secondary structure of proteins. With unlabelled protein the usual procedure is to record a set of two dimensional homonuclear nuclear magnetic resonance experiments through correlation spectroscopy (COSY), of which several types include conventional correlation spectroscopy and nuclear Overhauser effect spectroscopy (NOESY).¹ A two-dimensional nuclear magnetic resonance experiment produces a two-dimensional spectrum. The units of both axes are chemical shifts. The COSY transfers magnetization through the chemical bonds between adjacent protons. The conventional correlation spectroscopy experiment is only able to transfer magnetization between protons on adjacent atoms, so it is transferred among all the protons that are connected by adjacent atoms.

Thus in a conventional correlation spectroscopy, an alpha proton transfers magnetization to the beta protons, the beta protons transfers to the alpha and gamma protons, if any are present, then

experiment is used to build so called spin systems, that is build a list of resonances of the chemical shift of the peptide proton, the alpha protons and all the protons from each residue’s side chain. Which chemical shifts corresponds to which nuclei in the spin system is determined by the conventional correlation spectroscopy connectivities and the fact that different types of protons have characteristic chemical shifts. To connect the different spin systems in a sequential order, the nuclear Overhauser effect spectroscopy experiment has to be used. Because this experiment transfers magnetization through space, it will show crosspeaks for all protons that are close in space regardless of whether they are in the same spin system or not. The neighbouring residues are inherently close in space, so the assignments can be made by the peaks in the NOESY with other spin systems.

1.6.4 Hydrogen deuterium exchange and variation of temperature

This two different studies allows to identify which protons of a molecule are involved in an intramolecular hydrogen bond. In the case of the hydrogen deuterium exchange, to a peptide solved in a solvent which does not present exchangeable deuterium will be added CD₃OD. The amide protons in the protein exchange readily with the deuterium of the solvent, so the hydrogen deuterium exchange by NMR spectroscopy follows the disappearance of the amide signals. How rapidly a given amide exchanges reflects its solvent accessibility. Thus amide exchange rates can give information on which parts of the protein are buried, hydrogen bonded etc.

The same principle is at the base of the variation of temperature studies. Generally it is accepted that an amide proton involved in a strong intramolecular hydrogen bond has a low temperature dependence coefficient (∆δ<3 ppb/K), while for a weak intramolecular hydrogen bond is significantly higher (∆δ>8 ppb/K).

1.6.5 Circular dichroism

Circular dichroism (CD) is a form of spectroscopy based on the differential absorption of left- and right-handed circularly polarized light. It can be used to help to determine the structure of macromolecules (including the secondary structure of proteins and the handedness of DNA). CD

solution in high dilution (typically <1 mM, to avoid peptide aggregation) in the absorption band of amide bonds (180-250 nm). Circular dichroism measures the ellipticity of a peptide in this band with UV polarised light. In this band can be observed the absorption π→π* of amides and the absorption will vary according to the hydrogen bonded or non hydrogen bonded state of the amides, therefore it will give information on the presence of a secondary structure. Indeed, a peptide being a chiral molecule, it will present an optical rotation on polarised light but this optical rotation can vary with the wavelength. The analysis is often performed in quartz cells of 1 mm length or less as the solvent absorption can create some parasite noise. All solvents can not be used in CD spectroscopy as the circular dichroism must be measured in a band where the solvent does not absorb, methanol (limit at 195 nm for a cell 1 mm long), trifluoroethanol (TFE) or mixtures methanol/water can be used. Other common organic solvents as THF, acetonitrile, chloroform or dichloromethane can not be used in this case as they absorb in the same region as amides.

The ellipticity θ follows the Beer-Lambert law and can be calculated namely:

θ = CD_measured/(Cxlxn)

with θ: ellipticity in deg.cm².dmol^-1 C. concentration in mol.L^-1

l: length of the cell in dm

n: number of NH in the molecule

This technique has the advantage to be fast and one can see almost immediately if the peptide adopts a secondary structure or not. The limitation is that one can not deduce exactly which amides are involved in hydrogen bonding. It is nevertheless very useful since CD curves of α-peptides are typical of a certain secondary structure and so new peptide curves can be confronted with references. There has been also a lot of work in the field of β-peptides and some references are available but when a peptide containing unnatural amino acids is analysed, this comparison can not be always done with certainty as the curves may differ a lot for a same secondary structure.

1.6.6 IR in solution

Another powerful tools to detect the presence of intramolecular hydrogen is the IR in solution.

The technique consist in the measurement of an IR spectrum at high dilution (usually in dichloromethane or chloroform) to avoid the peptide aggregation. It is so observed the region of amide bond, which present, in the case of intramolecular hydrogen bond, two different bands, one at less than 3400 cm^-1 bonded amides and another at more than 3400 cm^-1 for non bonded amide.

Limits of this technique are the impossibility to use solvent which can form hydrogen bond with the peptide or which adsorb in the interesting region (DMSO, methanol, etc.).

1.6.7 X-ray crystallography

This is the most potent tools to directly study the secondary conformation of a peptide.

Unfortunately, it is not simple to obtain a crystal of a peptide, especially for the high flexible linear ones. Actually, the crystal structure of more and more peptides of biological interest are available on the Protein Data Bank database.