Question

What is the experimental evidence that proteins fold into compact, well-ordered structures?

What is diffraction? How is diffraction used to determine the three-dimensional fold of a protein?

Answer 1

I would like to point out SAXS as technique that can provide structural information and the overall shape of your protein. While SAXS will tell you if your protein or parts of it are folded, it can't provide information on discrete secondary structure elements (which CD can provide). On the other hand, CD can't provide you with an overall shape of your protein.

SAXS can tell you the overall size and shape of your protein molecule in solution and it can give you an indication of disorder (if the overall molecule or certain domains/regions are disordered). If your protein is very stable with rigid domains (as opposed to domains joined by floppy linkers) it can also give you a good low resolution structure of the whole molecule. The resolution of a SAXS structure is comparable to a high quality 3D EM structure. This is obviously not as informative as an X-ray or NMR structure, but it's good for interaction studies, showing which parts of a protein molecule are involved in forming multimers (dimers, trimers. etc...) or how it interacts with a binding partner (eg. which part of protein A binds to which part of protein B). SAXS also has the advantage that it is quick and easy and there's no need for the expensive protein labelling needed for NMR or for the expense, time and complicated procedures needed for growing protein crystals and solving X-ray structures.

Diffraction is the tendency of a beam to spread or bend as it propagates. This can be exaggerated if the beam passes through a slit or by some other obstacle. It is important to realize that diffraction is the thing that happens to the beam after it passes by the obstacle.

Diffraction plays a important role in xray crystallography ,when  a beam of x-rays of wavelength 1.54 Å is produced by accelerating electrons against a copper target. A narrow beam of x-rays strikes the protein crystal. Part of the beam goes straight through the crystal; the rest is scattered in various directions.diffracted, x-rays are detected by x-ray film, the blackening of the emulsion being proportional to the intensity of the scattered x-ray beam, or by a solid-state electronic detector. The scattering pattern provides abundant information about protein structure.