A new computer program (CORE) is described that predicts core hydrophobic sequences of predetermined target protein structures. A novel scoring function is employed, which for the first time incorporates parameters directly correlated to free energies of unfolding (ΔGu), melting temperatures (Tm), and cooperativity. Metropolis-driven simulated annealing and low-temperature Monte Carlo sampling are used to optimize this score, generating sequences predicted to yield uniquely folded, stable proteins with cooperative unfolding transitions. The hydrophobic core residues of four natural proteins were predicted using CORE with the backbone structure and solvent exposed residues as input. In the two smaller proteins tested (Gβ1, 11 core amino acids; 434 cro, 10 core amino acids), the native sequence was regenerated as well as the sequence of known thermally stable variants that exhibit cooperative denaturation transitions. Previously designed sequences of variants with lower thermal stability and weaker cooperativity were not predicted. In the two larger proteins tested (myoglobin, 32 core amino acids; methionine aminopeptidase, 63 core amino acids), sequences with corresponding side-chain conformations remarkably similar to that of native were predicted.

α1-Antitrypsin deficiency, which can lead to both emphysema and liver disease, is a result of the accumulation of α1-antitrypsin polymers within the hepatocyte. A wealth of biochemical and biophysical data suggests that α1-antitrypsin polymers form via insertion of residues from the reactive center loop of one molecule into the β-sheet of another. However, this long-standing hypothesis has not been confirmed by direct structural evidence. Here, we describe the first crystallographic evidence of a β-strand linked polymer form of α1-antitrypsin: the crystal structure of a cleaved α1-antitrypsin polymer.

Proline peptide group isomerization can result in kinetic barriers in protein folding. In particular, the cis proline peptide conformation at Tyr92–Pro93 of bovine pancreatic ribonuclease A (RNase A) has been proposed to be crucial for chain folding initiation. Mutation of this proline-93 to alanine results in an RNase A molecule, P93A, that exhibits unfolding/refolding kinetics consistent with a cis Tyr92–Ala93 peptide group conformation in the folded structure (Dodge RW, Scheraga HA, 1996, Biochemistry 35:1548–1559). Here, we describe the analysis of backbone proton resonance assignments for P93A together with nuclear Overhauser effect data that provide spectroscopic evidence for a type VI β-bend conformation with a cis Tyr92–Ala93 peptide group in the folded structure. This is in contrast to the reported X-ray crystal structure of [Pro93Gly]-RNase A (Schultz LW, Hargraves SR, Klink TA, Raines RT, 1998, Protein Sci 7:1620–1625), in which Tyr92–Gly93 forms a type-II β-bend with a trans peptide group conformation. While a glycine residue at position 93 accommodates a type-II bend (with a positive value of φ93), RNase A molecules with either proline or alanine residues at this position appear to require a cis peptide group with a type-VI β-bend for proper folding. These results support the view that a cis Pro93 conformation is crucial for proper folding of wild-type RNase A.

A novel method for monitoring fibrillogenesis is developed and applied to the amyloidogenic peptide, islet amyloid polypeptide (IAPP). The approach, based on electrospray ionization mass spectrometry, is complementary to existing assays of fibril formation as it monitors directly the population of precursor rather than product molecules. We are able to monitor fiber formation in two modes: a quenched mode in which fibril formation is halted by dilution into denaturant and a real time mode in which fibril formation is conducted within the capillary of the electrospray source. Central to the method is the observation that fibrillar IAPP does not compromise the ionization of monomeric IAPP. Furthermore, under mild ionization conditions, fibrillar IAPP does not dissociate and contribute to the monomeric signal. Critically, we introduce an internal standard, rat IAPP, for analysis on the mass spectrometer. This standard is sufficiently similar in sequence in that it ionizes identically to human IAPP. Furthermore, the sequence is sufficiently different in that it does not form fibrils and is distinguishable on the basis of mass. Applied to IAPP fibrillogenesis, our technique reveals that precursor consumption in seeded reactions obeys first-order kinetics. Furthermore, a consistent level of monomer persists in both seeded and unseeded experiments after the fibril formation is complete. Given the inherent stability of fibrils, we expect this approach to be applicable to other amyloid systems.