Sir Lawrence Bragg.

An extract from the obituary for Sir Lawrence Bragg by M. F. Perutz [Nature (London), (1971), 233, 74-76] is given.

Amyloid fibers are water-filled nanotubes.

A study of papers on amyloid fibers suggested to us that cylindrical beta-sheets are the only structures consistent with some of the x-ray and electron microscope data. We then found that our own 7-year-old and hitherto enigmatic x-ray diagram of poly-L-glutamine fits a cylindrical sheet of 31 A diameter made of beta-strands with 20 residues per helical turn. Successive turns are linked by hydrogen bonds between both the main chain and side chain amides, and side chains point alternately into and out of the cylinder. Fibers of the exon-1 peptide of huntingtin and of the glutamine- and asparagine-rich region of the yeast prion Sup35 give the same underlying x-ray diagrams, which show that they have the same structure. Electron micrographs show that the 100-A-thick fibers of the Sup35 peptide are ropes made of three protofibrils a little over 30 A thick. They have a measured mass of 1,450 Da/A, compared with 1,426 Da/A for a calculated mass of three protofibrils each with 20 residues per helical turn wound around each other with a helical pitch of 510 A. Published x-ray diagrams and electron micrographs show that fibers of synuclein, the protein that forms the aggregates of Parkinson disease, consist of single cylindrical beta-sheets. Fibers of Alzheimer A beta fragments and variants are probably made of either two or three concentric cylindrical beta-sheets. Our structure of poly-L-glutamine fibers may explain why, in all but one of the neurodegenerative diseases resulting from extension of glutamine repeats, disease occurs when the number of repeats exceeds 37-40. A single helical turn with 20 residues would be unstable, because there is nothing to hold it in place, but two turns with 40 residues are stabilized by the hydrogen bonds between their amides and can act as nuclei for further helical growth. The A beta peptide of Alzheimer's disease contains 42 residues, the best number for nucleating further growth. All these structures are very stable; the best hope for therapies lies in preventing their growth.

Aggregation of proteins with expanded glutamine and alanine repeats of the glutamine-rich and asparagine-rich domains of Sup35 and of the amyloid beta-peptide of amyloid plaques.

The exon-1 peptide of huntingtin has 51 Gln repeats and produces the symptoms of Huntington's disease in transgenic mice. Aggregation of the yeast Sup35 protein into prions has been attributed to its glutamine-rich and asparagine-rich domain. Here, we show that poly-L-asparagine forms polar zippers similar to those of poly-L-glutamine. In solution at acid pH, the glutamine-rich and asparagine-rich 18-residue Sup35 peptide, rendered soluble by the addition of two aspartates at the amino end and two lysines at the carboxyl end, gives a beta-sheet CD spectrum; it aggregates at neutral pH. A poly-alanine peptide D(2)A(10)K(2) gives an alpha-helical CD spectrum at all pHs and does not aggregate; a peptide with the sequence of the C-terminal helix of the alpha-chain of human hemoglobin, preceded by two aspartates and followed by two lysines, exhibits a random coil spectrum and does not aggregate either. Alignment of several beta-strands with the sequence of the 42-residue Alzheimer's amyloid beta-peptide shows that they can be linked together by a network of salt bridges. We also asked why single amino acid replacements can so destabilize the native structures of proteins that they unfold and form amyloids. The difference in free energy of a protein molecule between its native, fully ordered structure and an amorphous mixture of randomly coiled chains is only of the order of 10 kcal/mol. Theory shows that destabilization of the native structure by no more than 2 kcal/mol can increase the probability of nucleation of disordered aggregates from which amyloids could grow 130,000-fold.

Cause of neural death in neurodegenerative diseases attributable to expansion of glutamine repeats.

Neurodegenerative diseases resulting from expanded repeat sequences of glutamine residues are associated with the formation of protein aggregates in the cell nuclei of the affected neurons, but whether these are pathogenic is controversial. Recent observations indicate that the ages of onset of these diseases are exponential functions of the repeat lengths and that the probability of neural death is constant with time. The only process known to us that could give rise to such behaviour is nucleation of the aggregates.

Fix L, a haemoglobin that acts as an oxygen sensor: signalling mechanism and structural basis of its homology with PAS domains.

Fix L, which contains a haemoglobin domain homologous to the PAS family and a histidine kinase domain, forms, with Fix J, a two-component signalling complex that regulates expression of nitrogenase genes in Rhizobium. Spin transitions of its haem iron trigger stereochemical changes in and around the haem that, together with steric effects, control the activity of the kinase. Homology with the PAS family is based on a common core of about 20 structurally equivalent sites from which polar residues are excluded.

Glutamine repeats and neurodegenerative diseases: molecular aspects.

Eight severe inherited neurodegenerative diseases are caused by expansion of glutamine repeats in the affected proteins. In every case, proteins with repeats of fewer than 38 glutamine residues are harmless, but those with repeats of more than 41 glutamine residues form toxic neuronal nuclear aggregates in the affected neurons. Similarly, proteins that have repeats of fewer than 37 glutamine residues are soluble in vitro, whereas proteins with repeats of more than 40 glutamine residues precipitate as insoluble fibres, apparently because of a structural transition associated with the increased length.

Crystal structure of a dimeric chymotrypsin inhibitor 2 mutant containing an inserted glutamine repeat.

We have constructed mutants of chymotrypsin inhibitor 2 with short glutamine repeats inserted into its inhibitory loop. These mutants oligomerize when expressed in Escherichia coli. The dimer of a mutant with four glutamines now has been crystallized, and its structure has been solved by molecular replacement by using the wild-type monomer as a search model. The structure of each half of the dimer is found to be the same as that of the wild-type monomer, except around the glutamine insertion. It was proposed that the components of the oligomers are held together by hydrogen bonds between the main-chain and side-chain amides of the glutamine repeats. Instead, they appear to form by swapping domains on folding in E. coli, and the glutamine repeats connecting the components of the dimers are disordered.

Allosteric mechanism of haemoglobin: rupture of salt-bridges raises the oxygen affinity of the T-structure.

The T-structure of human haemoglobin is linked by salt-bridges between its four subunits, formed by the C-terminal arginine residues of the alpha-subunits and the C-terminal histidine residues of the beta-subunits. In the R-structure, these salt-bridges are absent. The oxygen affinity of the T-structure is lower than that of the R-structure by the equivalent of 3.5 kcal/mol haem. This difference has been attributed to the constraints imposed upon the T-structure by the salt-bridges, which were thought to hinder the changes in tertiary structure needed for firm oxygen binding. We have subjected this postulate to a rigorous test by measuring the oxygen equilibria of T-state crystals of an abnormal human haemoglobin in which the C-terminal histidine residues of the beta-chains are replaced by leucine residues. This replacement removes the salt-bridges from the histidine imidazole groups to the neighbouring aspartate residues. The crystals have an oxygen affinity about three times greater than that of crystals of normal haemoglobin. Hill's coefficient is close to unity. The oxygen affinity is unaffected by pH, chloride or the allosteric effector bezafibrate. Equilibrium curves determined by single crystal microspectrophometry using light polarised parallel and normal to the crystallographic a-axis show no significant difference between the oxygen affinities of alpha and beta-haems. Our results show that rupture of salt-bridges raises the oxygen affinity of the T-structure even when this is clamped firmly by the crystal lattice.

The stereochemical mechanism of the cooperative effects in hemoglobin revisited.

In 1970, Perutz tried to put the allosteric mechanism of hemoglobin, proposed by Monod, Wyman and Changeux in 1965, on a stereochemical basis. He interpreted their two-state model in terms of an equilibrium between two alternative structures, a tense one (T) with low oxygen affinity, constrained by salt-bridges between the C-termini of the four subunits, and a relaxed one (R) lacking these bridges. The equilibrium was thought to be governed primarily by the positions of the iron atoms relative to the porphyrin: out-of-plane in five-coordinated, high-spin deoxyhemoglobin, and in-plane in six-coordinated, low-spin oxyhemoglobin. The tension exercised by the salt-bridges in the T-structure was to be transmitted to the heme-linked histidines and to restrain the movement of the iron atoms into the porphyrin plane that is necessary for oxygen binding. At the beta-hemes, the distal valine and histidine block the oxygen-combining site in the T-structure; its tension was thought to strengthen that blockage. Finally, Perutz attributed the linearity of proton release with early oxygen uptake to the sequential rupture of salt-bridges in the T-structure and to the accompanying drop in pKa of the weak bases that form part of them. Almost every feature of this mechanism has been disputed, but evidence that has come to light more than 25 years later now shows it to have been substantially correct. That new evidence is reviewed below.

Long live the Queen's subjects.

In 1952, the Queen congratulated 255 people on their hundredth birthdays and 1135 couples on their sixtieth wedding anniversaries. By 1996, these numbers had risen to 5218 and 11,688, respectively. Semilogarithmic plots, normalized to constant numbers of births and marriages, show steady exponential rises in the number of centenarians with a doubling time of 11 years, and of diamond weddings with a doubling time of 19 years. An alternative plot of the numbers of those reaching a hundred between 1910 and 1990, based on registers of births and deaths and normalized to constant births, shows an annual rise of only 1% from 1910 to 1946, followed by a steady exponential rise with a doubling time of 12 years, closely matching that of 11 years derived from the Queen's figures. The exponential rise in the number of those born from 1846 onwards living to a hundred precedes by many years the general rise in the expectation of life at birth and the general drop in mortality from infectious diseases, but it coincides with the beginning of a steady rise in real wages. Another important factor may be improved medical treatment at old age from 1946 onwards.

Glutamine repeats and inherited neurodegenerative diseases: molecular aspects.

Several dominantly inherited, late onset, neurodegenerative diseases are due to expansion of CAG repeats, leading to expansion of glutamine repeats in the affected proteins. These proteins are of very different sizes and, with one exception, show no sequence homology to known proteins or to each other; their functions are unknown. In some, the glutamine repeat starts near the N-terminus, in another near the middle and in another near the C-terminus, but regardless of these differences, no disease has been observed in individuals with fewer than 37 repeats, and absence of disease has never been found in those with more than 41 repeats. Protein constructs with more than 41 repeats are toxic to E. coli and to CHO cells in culture, and they elicit ataxia in transgenic mice. These observations argue in favour of a distinct change of structure associated with elongation beyond 37-41 glutamine repeats. The review describes experiments designed to find out what these structures might be and how they could influence the properties of the proteins of which they form part. Poly-L-glutamines form pleated sheets of beta-strands held together by hydrogen bonds between their amides. Incorporation of glutamine repeats into a small protein of known structure made it associate irreversibly into oligomers. That association took place during the folding of the protein molecules and led to their becoming firmly interlocked by either strand- or domain-swapping. Thermodynamic considerations suggest that elongation of glutamine repeats beyond a certain length may lead to a phase change from random coils to hydrogen-bonded hairpins. Possible mechanisms of expansion of CAG repeats are discussed in the light of looped DNA model structures.

Cause of the root effect in fish haemoglobins.

The unusual properties of the Root effect haemoglobins in teleost fish --which allow them to pump O2 into their swim bladders and eyes against very high pressures--are illuminated in a new fish haemoglobin structure.