Question

1. we discussed a classification scheme of amino acids that divided them into three groups based on Polarity. what are the three groups of amino acids and where are you most likely to find amino acid residues belonging to each group within a protein?

2. what are the benefits and pitfalls of each method of protein structure determination?

Answer 1

1. Based on Polarity following three groups can be formed:

Polar Amino Acids with no Charge:

These amino acids do not have any charge on the 'R' group. These amino acids participate in hydrogen bonding of protein structure. The amino acids in this group are serine, threonine, tyrosine, cysteine, glutamine and aspargine.

Polar Amino Acids with Positive Charge:

Polar amino acids with positive charge have more amino groups as compared to carboxyl groups making it basic. The amino acids, which have positive charge on the 'R' group are placed in this category. They are lysine, arginine and histidine.

Polar Amino Acids with Negative Charge:

Polar amino acids with negative charge have more carboxyl groups than amino groups making them acidic. The amino acids, which have negative charge on the 'R' group are placed in this category. They are called as dicarboxylic mono-amino acids. They are aspartic acid and glutamic acid.

2. Several methods are currently used to determine the structure of a protein, including X-ray crystallography, NMR spectroscopy, and electron microscopy. Each method has advantages and disadvantages.

X-ray Crystallography

Most of the structures included in the PDB archive were determined using X-ray crystallography. For this method, the protein is purified and crystallized, then subjected to an intense beam of X-rays. The proteins in the crystal diffract the X-ray beam into one or another characteristic pattern of spots, which are then analyzed (with some tricky methods to determine the phase of the X-ray wave in each spot) to determine the distribution of electrons in the protein. The resulting map of the electron density is then interpreted to determine the location of each atom. The PDB archive contains two types of data for crystal structures. The coordinate files include atomic positions for the final model of the structure, and the data files include the structure factors (the intensity and phase of the X-ray spots in the diffraction pattern) from the structure determination. X-ray crystallography can provide very detailed atomic information, showing every atom in a protein or nucleic acid along with atomic details of ligands, inhibitors, ions, and other molecules that are incorporated into the crystal. However, the process of crystallization is difficult and can impose limitations on the types of proteins that may be studied by this method. For example, X-ray crystallography is an excellent method for determining the structures of rigid proteins that form nice, ordered crystals. Flexible proteins, on the other hand, are far more difficult to study by this method because crystallography relies on having many, many molecules aligned in exactly the same orientation, like a repeated pattern in wallpaper. Flexible portions of protein will often be invisible in crystallographic electron density maps, since their electron density will be smeared over a large space.

Exploring Biological Structure and Function using X-ray Free Electron Lasers (XFEL)

New technology, termed serial femtosecond crystallography, is revolutionizing the methods of X-ray crystallography. A free electron X-ray laser (XFEL) is used to create pulses of radiation that are extremely short (lasting only femtoseconds) and extremely bright. A stream of tiny crystals (nanometers to micrometers in size) is passed through the beam, and each X-ray pulse produces a diffraction pattern from a crystal, often burning it up in the process. A full data set is compiled from as many as tens of thousands of these individual diffraction patterns. The method is very powerful because it allows scientists to study molecular processes that occur over very short time scales, such as the absorption of light by biological chromophores.

NMR Spectroscopy

NMR spectroscopy may be used to determine the structure of proteins. The protein is purified, placed in a strong magnetic field, and then probed with radio waves. A distinctive set of observed resonances may be analyzed to give a list of atomic nuclei that are close to one another, and to characterize the local conformation of atoms that are bonded together. This list of restraints is then used to build a model of the protein that shows the location of each atom. The technique is currently limited to small or medium proteins, since large proteins present problems with overlapping peaks in the NMR spectra.

A major advantage of NMR spectroscopy is that it provides information on proteins in solution, as opposed to those locked in a crystal or bound to a microscope grid, and thus, NMR spectroscopy is the premier method for studying the atomic structures of flexible proteins. A typical NMR structure will include an ensemble of protein structures, all of which are consistent with the observed list of experimental restraints. The structures in this ensemble will be very similar to each other in regions with strong restraints, and very different in less constrained portions of the chain. Presumably, these areas with fewer restraints are the flexible parts of the molecule, and thus do not give a strong signal in the experiment.