Proteins large, solution structures

Most of the molecules introduced in this chapter are hydrophobic. Even those molecules that have been functionalized to improve water-solubility (for example, CCVJ and CCVJ triethyleneglycol ester 43, Fig. 14) contain large hydrophobic structures. In aqueous solutions that contain proteins or other macromolecules with hydrophobic regions, molecular rotors are attracted to these pockets and bind to the proteins. Noncovalent attraction to hydrophobic pockets is associated with restricted intramolecular rotation and consequently increased quantum yield. In this respect, molecular rotors are superior protein probes, because they do not only indicate the presence of proteins (similar to antibody-conjugated fluorescent markers), but they also report a constricted environment and can therefore be used to probe protein structure and assembly. 

NOESY NMR spectroscopy is a homonuclear two-dimensional experiment that identifies proton nuclei that are close to each other in space. If one has already identified proton resonances in one-dimensional NMR spectroscopy or by other methods, it is then possible to determine three dimensional structure through NOESY. For instance, it is possible to determine how large molecules such as proteins fold themselves in three-dimensional space using the NOESY technique. The solution structures thus determined can be compared with solid-state information on the same protein obtained from X-ray crystallographic studies. The pulse sequence for a simple NOESY experiment is shown in Figure 3.23 as adapted from Figure 8.12 of reference 19. 

The main advantage of NMR spectroscopy is its use with proteins in solution. In consequence, rather than obtaining a single three-dimensional structure of the protein, the final result for an NMR structure is a set of more or less overlying structures which fulfill the criteria and constraints given particularly by the NOEs. Typically, flexibly oriented protein loops appear as largely diverging structures in this part of the protein. Likewise, two distinct local conformations of the protein are represented by two differentiated populations of NMR structures. Conformational dynamics are observable on different time scales. The rates of equilibration of two (or more) substructures can be calculated from analysis of the line shape of the resonances and from spin relaxation times Tj and T2, respectively. 

Very low-frequency vibrations have been observed in proteins (e.g., Brown et al., 1972 Genzel et al., 1976), which must involve concerted motion of rather large portions of the structure. By choosing a suitable set of proteins to measure (preferably in solution), it should be possible to decide approximately what structural modes are involved. Candidates include helix torsion, coupled changes of peptide orientation in /3 strands, and perhaps relative motions of entire domains or subunits. These hypotheses should be tested, because the low-frequency vibrations probably reflect large-scale structural properties that would be very useful to know. 

Knowing that crystallographers study proteins in the crystalline state, you may be wondering if these molecules are altered when they crystallize, and whether the structure revealed by X rays is pertinent to the molecule s action in solution. Crystallographers worry about this problem also, and with a few proteins, it has been found that crystal structures are in conflict with chemical or spectroscopic evidence about the protein in solution. These cases are rare, however, and the large majority of crystal structures appear to be identical to the solution structure. Because of the slight possibility that crystallization will alter molecular structure, an essential part of any structure determination project is an effort to show that the crystallized protein is not significantly altered. 

Recall that stable protein crystals contain a large amount of both ordered and disordered water molecules. As a result, the proteins in the crystal are still in the aqueous state, subject to the same solvent effects that stabilize the structure in solution. Viewed in this light, it is less surprising that proteins retain their solution structure in the crystal. 

The crystal structure of native hen ovalbumin shows an intact reactive center loop in the form of an exposed a-helix of three turns that protrudes from the main body of the molecule on two peptide stalks. The ovalbumin structure includes four crystallographically independent ovalbumin molecules and the position of the helical reactive center loop relative to the protein core differs by 2-3 A between molecules. Although this shift is probably due to the different environments of the helices in the crystal lattice, it suggested that the reactive center loop is flexible in solution. Structural studies of serpins in various conformations have shown how the exceptional mobility of the serpin reactive center loop and their unique flexibility is essential for function. In contrary to inhibitory serpins, ovalbumin does not show evidence for a large conformational change following cleavage at its putative reactive center and appears to have lost the extreme mobility which is characteristic for its inhibitory ancestors. 

While this notion may conjure up visions of plastic materials it is important to remember that proteins and nucleic acids are also polymers. Many proteins form globular structures and, indeed, may interlock to encapsulate a large volume of space as exemplified by the coatings of capsid viruses. In a prebiotic world, polypeptides could have formed in aqueous solution through the sequential reaction of amino acids. The individual amino acids hydrogen bond donor and acceptor groups, amines, carbonyls and carboxylic acids, would all have helped to keep the molecules in solution. Once a polypeptide had formed, however, many of these would be unavailable as they became incorporated in the hydrogen bond network that formed the secondary and tertiary structure. This would result in a more hydrophobic surface for the protein capsule which would make an effective cell. 

Xu Y et al (2006) A new strategy for structure determination of large proteins in solution without deuteration. Nat Methods 3 931-937 PDBID 2H35... 

Katayev EA, Sessler JL, Khrustalev VN et al (2007) Synthetic model of the phosphate binding protein solid-state structure and solution-phase anion binding properties of a large oligopyr-rolic macrocycle. J Org Chem 72 7244—7252... 

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