Three peptides covering the sequence regions corresponding to the first two (CspB-1), the first three (CspB-2), and the last two (CspB-3) β-strands of CspB, the major cold shock protein of Bacillus subtilis, have been synthesized and analyzed for their conformations in solution and for their precipitation behavior. The peptides are nearly insoluble in water, but highly soluble in aqueous solutions containing 50% acetonitrile (pH 4.0). Upon shifts of the solvent condition toward lower or higher acetonitrile concentrations, the peptides all form fibrils resembling those observed in amyloid associated diseases. These fibrils have been identified and characterized by electron microscopy, binding of the dye congo red, and X-ray fiber diffraction. Characterization of the peptides in solution by circular dichroism and NMR spectroscopy shows that the formation of these fibrils does not require specific preformed secondary structure in the solution state species. While the majority of the soluble fraction of each peptide is monomeric and unstructured, different types of structures including α-helical, β-sheet, and random coil conformations are observed under conditions that eventually lead to fibril formation. We conclude that the absence of tertiary contacts under solution conditions where binding interactions between peptide units are still favorable is a crucial requirement for amyloid formation. Thus, fragmentation of a sequence, like partial chemical denaturation or mutation, can enhance the capacity of specific protein sequences to form such fibrils.

All proteins of Saccharomyces cerevisiae have been compared to determine how frequently segments from one protein are present in other proteins. Proteins that are recently evolutionarily related were excluded. The most frequently present protein segments are long, tandem repetitions of a single amino acid. For some of these segments, up to 14% of all proteins in the genome were found to have similar peptides within them. These peptide segments may not be functional protein domains. Although they are the most common shared feature of yeast proteins, their ubiquity and simplicity argue that their probable function may be to simply serve as spacers between other protein motifs.

We have studied the conformation of β-lactoglobulin in aqueous solution at room temperature over the pH range ∼2.0–9.0 using vibrational Raman optical activity (ROA). The ROA spectra clearly show that the basic up and down β-barrel core is preserved over the entire pH range, in agreement with other studies. However, from the shift of a sharp positive ROA band at ∼1268 to ∼1294 cm−1 on going from pH values below that of the Tanford transition, which is centered at pH ∼7.5, to values above, the Tanford transition appears to be associated with changes in the local conformations of residues in loop sequences possibly corresponding to a migration into the α-helical region of the Ramachandran surface from a nearby region. These changes may be related to those detected in X-ray crystal structures which revealed that the Tanford transition is associated with conformational changes in loops which form a doorway to the interior of the protein. The results illustrate how the ability of ROA to detect loop and turn structure separately from secondary structure is useful for studying conformational plasticity in proteins.

Nanoflow electrospray mass spectrometry was used to monitor the formation of protein heterodimers of HU proteins from Bacillus stearothermophilus and Bacillus subtilis. This has enabled us to analyze both thermodynamic and kinetic features associated with the dissociation of homodimeric HU proteins. The results obtained correlate well with the kinetics of the protein dissociation process and the free energy difference between homo- and heterodimeric species anticipated from other studies. We suggest that this approach will have general applicability in studying protein association and dissociation under near-equilibrium conditions and will be relevant to a wide range of biological systems.

David Phillips was an outstanding scientist, one of the founding fathers of structural biology and a wise and influential figure in science and government. He started his research career with work on X-ray intensity statistics, then moved to small molecule crystallography, followed by protein crystallography and instrument design. Protein crystallography led to proposals for structure/function relationships, homology modeling, fundamental understanding of thermal motion, and several new protein structures of pharmaceutical interest. However, it is for his work with lysozyme that he will be most widely remembered. In 1966, he and the team, working at the Royal Institution in London, solved the first structure of an enzyme, lysozyme: from the structure it was immediately possible to put forward proposals for catalytic activity. The work first showed the power of protein crystallography to explain biological function in terms of physics and chemistry. It opened the way to the explosion in the number of protein structures that are now being determined with modern technology, and for the insights that these structures provide for the benefit of fundamental research, medicine, and agriculture.