Structure to Elucidate Mechanism Detailed structural information can give unexpected insights. Classic example: the elucidation of the structure of DNA by Watson and Crick (1952). The structure immediately suggested a means by which DNA could be replicated in a way that maintained its informational content

When is it appropriate to analyze the structure of a molecule? to understand a mechanism in greater detail Structure based drug design Novel structure Conformational switches

Historically structure determination performed late in the process of elucidating the function of a molecule. That may be changing…. reliable means of predicting 3D structures from primary sequence? - “structural genomics” initiative to find the structures of large numbers of proteins.

Experimental prerequisites for obtaining a structure! Need a purified molecule often in abundance. Proteolysis helps to define a domain and then can overexpress in heterologous system and purify or can synthesize if segment is small enough. Helps to have sufficient knowledge of molecule to know how to produce a fragment on which to focus efforts. Need a lack of flexibility. For a crystal to form, must have a small number of conformations so that solution can get aggregation of similar conformations to grow crystal.

Pitfalls -biological relevance. Need to be thoughtful about interpretation. Again need to test theories.

What are some methods for analyzing structure??

Xray crystalography Circular dichroism (CD) NMR Sedimentation Atomic force microscopy Electronparametic spin resonance

XRAY Crystallography Identifies chrstalline phases present in solid materials. A beam of Xrays are used to bombard a specimen from various angles. The Xrays are diffracted as they are reflected from successive planes formed by the crystal lattice of the material. By varying angles a diffraction pattern emerges that is characteristic of the samples. It can then be further characterized by comparing with databases of other patterns. Heavy atoms with more electrons scattern X-rays more strongly.

Different methods for Xray crystallography MIR: multiple isomorphous replacement Depends upon the select binding of heavy metals (mercury/platnum) to a limit number of sites in the crystal. Heavy atoms scatter xrays more strongly than light atoms they replace MAD: multiwavelength anomalous diffraction. Replacement of methionine with selenomethionine by overexpressing the protein in bacteria grown in defined media containing selenomethionine. The selenium atom replaces a sulfur in the methionine side chain. Unlike larger metals used for MIR these selenium atoms are tolerated well by most proteins, crystallization tends to proceed the same as native protein. The selenium is readily detected by x-ray diffraction.

Protein models Electron density map What you predict End result What you see

Circular Dichroism Spectroscopy Circular dichroism (CD) spectroscopy measures differences in the absorption of left-handed polarized light versus right-handed polarized light which arise due to structural asymmetry. The absence of regular structure results in zero CD intensity, while an ordered structure results in a spectrum. Circular dichroism spectroscopy is particularly good for: -determining whether a protein is folded, and if so characterizing its secondary structure, tertiary structure, and the structural family to which it belongs -comparing the structures for different mutants of the same protein -studying the conformational stability of a protein under stress -- thermal stability, pH stability, and stability to denaturants. I

Example of how a particular structure appears in CD

Sedimentation equilibrium Sedimentation equilibrium is an analytical ultracentrifugation method  for measuring protein molecular masses in solution and for studying protein-protein interactions. Sample spun in ultracentrifuge to force the protein toward outside of rotor, but not high enough to cause the sample to pellet. As centrifugal force produces a gradient in protein concentration, diffusion acts to oppose this concentration gradient. An exact balance is reached between sedimentation and diffusion and the concentration distribution reaches equilibrium. This equilibrium concentration is then measured while the sample is spinning using absorbance detection. It is particularly valuable for: -establishing whether the native state of a protein is a monomer, dimer, trimer, etc. -measuring the stoichiometry of complexes between two or more different proteins or between a protein and a non-protein ligand -measuring the equilibrium constants for reversible protein-protein and protein-ligand interactions (Kd).

Example of trimers using sedimentation equilibrium

Mechanism for conformational change of influenza hemagglutinin Infection of cells by influenza virus (flu)

Model of the fusion between HA and membrane

Tubby Tub identified by positional cloning in obese mice Tub -/- display degeneration of retina, hearing loss TUBLP (Tubby like protein) in human: mutation Retinitis pigmentosa Molecular role unknown

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Model?

Other biophysics methods to address questions Regarding proteins (conformation, size…)

Figure 1

Figure 2

Figure 3

Figure 4: Model

Figure 5

Figure 6

Figure 7