Protein Structure-Function #6

Determination of Protein Structure and Protein Engineering

X-Ray Crystallography

 X-rays are a form of electromagnetic radiation with a wavelength typically around 0.1nm (the diameter of a hydrogen atom).
If a narrow parallel beam of x-rays is directed at a sample of pure protein, most of the x-rays pass straight through it. A small fraction will however be scattered by the atoms in the sample. If the sample is a well ordered crystal, the scattered waves reinforce one another at certain points and appear as diffraction spots when the x-rays are recorded by a suitable detector.

X-Ray Crystallography

 The position and intensity of each spot contains information about the locations of the atoms within the crystal that gave rise to it. Deducing the three dimensional structure of a large molecule form the diffraction pattern is a complicated task and was not achieved for a protein molecule until the 1960s. An electron density map is produced from the overall diffraction pattern using a mathematical technique called a Fourier transform. A model for the structure is then built that is consistent with the electron-density map.

Problems
In recent years X-ray diffraction has become increasingly automated, however many problems still remain with the technique. The slowest step is often the generation of suitable crystals. This requires large amounts of very pure protein and often involves many years of trial and error, searching for the proper crystallization conditions. There are still many proteins, in particular membrane proteins, which have so far resisted all attempts to crystallize them.

 The physical environment of the crystal is not identical with that within a living cell. A crystal imposes a space and time average on the structure deduced from the analysis and thus provides little information about molecular motion within the protein (e.g. calmodulin).
The conformation of the molecules within the crystal can also be affected by nonphsysiological factors such as incidental protein-protein interactions.

 Structures derived through this method should always be confirmed by comparing structural data acquired by other means. Also mobile portions of the protein are difficult to determine as they may have a different confirmation in different protein molecules within the crystal.

Nuclear Magnetic Resonance (NMR) Spectroscopy

This technique depends on the fact that certain atomic nuclei are intrinsically magnetic.
The simplest example is the hydrogen nucleus (1H), which is a proton.

The spinning of a proton generates a magnetic moment. This moment can take either of two orientations, or spin states (called and ), when an external magnetic field is applied.
The energy difference between these states is proportional to the strength of the imposed magnetic field.

 The state has a slightly lower energy and hence is slightly more populated (by a factor of the order of 1.00001 in a typical experiment) because it is aligned with the field. A spinning proton in an state can be raised to an excited state ( state) by applying a pulse of electromagnetic radiation (a radio-frequency, or RF, pulse), provided the frequency corresponds to the energy difference between the and the states.
In these circumstances, the spin will change from to ; in other words, resonance will be obtained.

 A resonance spectrum for a molecule can be obtained by varying the magnetic field at a constant frequency of electromagnetic radiation or by keeping the magnetic field constant and varying electromagnetic radiation.

 These properties can be used to examine the chemical surroundings of the hydrogen nucleus. The flow of electrons around a magnetic nucleus generates a small local magnetic field that opposes the applied field. The degree of such shielding depends on the surrounding electron density. Consequently, nuclei in different environments will change states, or resonate, at slightly different field strengths or radiation frequencies. The nuclei of the perturbed sample absorb electromagnetic radiation at a frequency that can be measured.

 The different frequencies, termed chemical shifts, are expressed in fractional units (parts per million, or ppm) relative to the shifts of a standard compound, such as a water-soluble derivative of tetramethysilane, that is added with the sample. For example, a -CH3 proton typically exhibits a chemical shift () of 1 ppm, compared with a chemical shift of 7 ppm for an aromatic proton.

Increasing Complexity

 It is possible to resolve most protons in many proteins by using this technique of onedimensional NMR. With this information, we can then deduce changes to a particular chemical group under different conditions, such as the conformational change of a protein from a disordered structure to an helix in response to a change in pH.

NOESY
We can garner even more information by examining how the spins on different protons affect their neighbors. By inducing a transient magnetization in a sample through the application a radiofrequency pulse, it is possible to alter the spin on one nucleus and examine the effect on the spin of a neighbouring nucleus.

NOESY
Especially revealing is a two-dimensional spectrum obtained by Nuclear Overhauser Enhancement Spectroscopy (NOESY), which graphically displays pairs of protons that are in close proximity, even if they are not close together in the primary structure

 The basis for this technique is the nuclear Overhauser effect (NOE), an interaction between nuclei that is proportional to the inverse sixth power of the distance between them.
Magnetization is transferred from an excited nucleus to an unexcited one if they are less than about 5 apart. In other words, the effect provides a means of detecting the location of atoms relative to one another in the three-dimensional structure of the protein. The diagonal of a NOESY spectrum corresponds to a one-dimensional spectrum. The off-diagonal peaks provide crucial new information: they identify pairs of protons that are less than 5 apart

(A) An example of the data from an NMR machine. This two-dimensional NMR spectrum is derived from the C-terminal domain of the enzyme cellulase. The spots represent interactions between hydrogen atoms that are near neighbors in the protein and hence reflects the distance that separates them. Complex computing methods, in conjunction with the known amino acid sequence, enable possible compatible structures to be derived. (B) In (B) 10 structures, which all satisfy the distance constraints equally well, are shown superimposed on one another, giving a good indication of the probable three-dimensional structure

Why So Many Structures?

A family of related structures is generated for three reasons. First, not enough constraints may be experimentally accessible to fully specify the structure.
Second, the distances obtained from analysis of the NOESY spectrum are only approximate.

Finally, the experimental observations are made not on single molecules but on a large number of molecules in solution that may have slightly different structures at any given moment. Thus, the family of structures generated from NMR structure analysis indicates the range of conformations for the protein in solution.

Problems?
At present, NMR spectroscopy can determine the structures of only relatively small proteins (<40 kd), but its resolving power is certain to increase.
The power of NMR has been greatly enhanced by the ability to produce proteins labeled uniformly or at specific sites with 13C, 15N, and 2H with the use of recombinant DNA technology

Circular Dichroism Spectroscopy

Protein dynamics and secondary structure determination

Circular Dichroism Spectroscopy

Circular dichroism (CD) spectroscopy measures differences in the absorption of left-handed polarized light versus right-handed polarized light that arise due to structural asymmetry. (measured as a quantity called mean residue whose units are degrees cm2dmol-1)

CD Spectroscopy
The absence of regular structure results in zero CD intensity, while an ordered structure results in a spectrum which can contain both positive and negative signals. Chiral or asymmetric molecules produce a CD spectrum because they absorb left and right handed polarized light to different extents and are thus considered to be optically active.
Biological macromolecules such as proteins and DNA are composed of optically active elements and because they can adopt different types of 3D structures, each type of molecule produces a distinct CD spectrum.

CD Spectroscopy

Different types of protein secondary structures (helices, sheets turns and coils) give rise to different CD spectra.
Because the spectrum of a protein is directly related to its secondary structure content, the spectrum will be the linear combination of each of these reference spectra, weighted by the fraction of the type of secondary structure. Therefore it is possible to mathematically extract the secondary structure information for an unknown protein from its CD spectra

CD Spectroscopy Has Been Used to Monitor

Secondary structure
Conformational changes Environmental effects

Protein folding and denaturation Dynamics

CD Spectrum of Recombinant IMMUTANS

 Some of the advantages of CD spectroscopy are that it requires only very small amounts of protein (100mg or less)

and measurement can be done very quickly (30 mins or less).

But this technique will not allow the 3D structure to be determined.

 Single Particle Cryo- EM

Visualizing biological complexes such as viruses, small organelles, and macromolecular biological complexes of 200 kDa or larger preserved in noncrystaline ice

Cryo-EM analysis 31 resolution

GroEL-GroES Complex (Cpn60)

Structure of the S. cerivisiae ATPase at 24 in the detergent Brij-35

Protein Engineering
1. Dissection of the structure and activity of existing proteins by making systematic alterations of their structures and examining the changes in their properties. 2. Production of novel proteins for use in medicine and industry.
3. We do not know how to design functional protein de novo, but we can make minor alterations of existing proteins to give useful changes in activity. We can also cut and paste segments of one protein to another. A good example is the humanization of monoclonal antibodies by grafting the mouse antigen-binding loops onto human frame work allowing use in cancer treatments.

 The prerequisite for protein engineering studies is that the enzyme has been cloned an expressed. Further, unless only relatively crude information is required, it is essential that the structure has been solved at high resolution

Choice of Mutation
Choose a mutation that deletes part of a side chain or leads to an isosteric change. Deletions are preferred to mutations that increase the size of the side chain, especially in the interior of a protein or at an enzyme-substrate interface. Any increase in volume of the side chain is liable to distort the structure of the enzyme whereas small changes in the enzyme cavity can be tolerated.

Avoid creating buried unpaired charges. Removal of a group that solvates a buried charge is dangerous. Solvation energies of ions are so high that charged groups must be solvated. Unless there is open access of solvent to buried charge residues, the structure of mutant proteins will rearrange so that there is solvation by water or other groups on the enzyme.

Choice of Mutation
Delete the minimal number of interactions. It is difficult enough to analyze the change in just one interaction. Avoid the deletion of multiple interactions where possible.
Do not add new functional groups to side chains. The addition of a new group on a side chain can cause local reorganization of structure if a new group can make novel interactions

All previous rules may be disobeyed when appropriate!

Choice of Mutation
The ideal mutation is a nondisruptive deletion, which is one which simply removes an interaction without causing a disruption or reorganization of a structure.
When in doubt mutate to alanine. This is generally a deletion mutation and alanine has few quirks. Glycine, because of it wider freedom of conformation can have odd effects on structure

Random Mutagenesis
The process of random mutagenesis is also useful in the study of protein structure function. Random mutations may be induced by PCR or by chemical mutagenesis. This process can be useful in identifying important residues when detailed structural information is not available.

Mutagenesis may also be performed at the gross level

Instead of changing a single amino acid residue, whole sections of the protein may be removed by excising a stretch of the gene. This is termed deletion mutagenesis. Domains of proteins may be spliced together to give novel proteins.
Groups can also be added to proteins to act as tags or as aids in purification process e.g. His tags or thioredoxin.