Craig J. Marshall

Cold-adapted enzymes

It is an article of faith among biochemists and molecular biologists that precious enzymes must be stored on ice. The usual reason given is that, at temperatures around freezing, enzyme activity is minimized and protein stability maximized. There is considerable evidence supporting this, but is it true for all enzymes? What about enzymes from organisms that spend part or all of their lives at temperatures around freezing? How do they manage to maintain normal enzymatic function at low temperatures? Can we learn something from cold-adapted proteins that would allow us better to understand how proteins function?

Freezing survival and cryoprotective dehydration as cold tolerance mechanisms in the Antarctic nematode Panagrolaimus davidi.

SUMMARY The relative importance of freezing tolerance and cryoprotective dehydration in the Antarctic nematode Panagrolaimus davidi has been investigated. If nucleation of the medium is initiated at a high subzero temperature (-1°C), the nematodes do not freeze but dehydrate. This effect occurs in deionised water, indicating that the loss of water is driven by the difference in vapour pressure of ice and supercooled water at the same temperature. If the nematodes are held above their nucleation temperature for a sufficient time, or are cooled slowly, enough water is lost to prevent freezing (cryoprotective dehydration). However, if the medium is nucleated at lower temperatures or if the sample is cooled at a faster cooling rate, the nematodes freeze and can survive intracellular ice formation. P. davidi thus has a variety of mechanisms that ensure its survival in its harsh terrestrial Antarctic habitat.

Laws of form revisited

efore Darwin, most biologists adheredto a platonic model of nature. Thisimplied that the biological realm con-sisted of a finite set of essentially immutablenatural forms that, like inorganic formssuch as atoms or crystals, are an intrinsicpart of the eternal order of the world. Justas, today, we account for the form of atomsand crystals by a set of physical laws or‘constructional rules’, so pre-darwinianbiologists sought to account for the originof biological forms in terms of a set ofgenerative physical laws often referred to asthe ‘laws of form’.For many biologists today, platonicbiology is an anachronism irretrievably laidto rest, and the idea that biological formsmight be intrinsic features of nature generat-ed by physical laws is treated with increduli-ty. However, recent advances in proteinchemistry suggest that at least one set ofbiological forms — the basic protein folds —is determined by physical laws similar tothose giving rise to crystals and atoms. Theygive every appearance of being invariantplatonic forms of precisely the type that thepre-darwinian biologists were seeking. Protein folds, the basic constructionalunits of proteins, each consist of a foldedchain of between 80 and 200 amino acids.Some proteins consist of a single fold, butmost are a combination of two or more.During the 1970s, as the three-dimensionalstructure of an increasing number of foldswas determined, it became apparent thatthe folds could be classified into a finitenumber of distinct structural families con-taining a number of closely related forms.The fact that protein folds could be classifiedin this manner provided the first line ofevidence that the folds might be naturalforms.Further evidence that the folds doindeed represent a finite set of naturalforms is provided by detailed structuralstudies carried out over the past two decadeswhich have revealed that the structure ofthe folds can be accounted for by whatamounts to a set of ‘constructional rules’governing the way that the various sec-ondary structural motifs, such as a-helicesand b-sheets, can be combined and packedinto compact three-dimensional structures.One is inevitably reminded of the atom-building rules governing the assembly ofsubatomic particles into the 92 atoms of theperiodic table. Consideration of these ‘constructionallaws’ suggests that the total number ofpermissible folds is bound to be restrictedto a very small number — about 4,000,according to one estimate. Confirmationthat this is probably so is provided by a differ-ent type of estimate, based on the discoveryrate of new folds. Using this method,Cyrus Chothia of Britain’s Medical ResearchCouncil estimated that the total number offolds utilized by living organisms may not bemore than 1,000. Subsequent estimates havegiven figures of between 500 and 1,000.Whatever the final figure, the fact thatthe total number of folds represents a tinystable fraction of all possible polypeptideconformations, determined by the laws ofphysics, reinforces the notion that the folds,like atoms, represent a finite set of built-innatural forms.The robustness of the folds offers anoth-er clue. The fact that the folds can retaintheir native conformations in the face ofmultiple different sorts of short-term defor-mations caused by the molecular turbulenceof the cell, and in the face of extensive, long-term evolutionary changes in their amino-acid sequences, is precisely what would beexpected if they are natural forms, specifiedby physical law. Again, the fact that thesame fold can be specified by many differ-ent, apparently unrelated amino-acidsequences, suggesting multiple separate dis-coveries during the course of evolution, isfurther evidence that the folds are intrinsicfeatures of the order of nature. Finally, thefact that in many cases the same fold isadapted to very different biochemical func-tions is precisely what would be expected ifprotein functions are secondary adaptationsof a set of primary, immutable, naturalforms. If forms as complex as the protein foldsare intrinsic features of nature, might someof the higher architecture of life also be deter-mined by physical law? The robustness ofcertain cytoplasmic forms, for example thespindle apparatus and the cell form of ciliateprotozoans such as

DNA photorepair in echinoid embryos: effects of temperature on repair rate in Antarctic and non-Antarctic species

SUMMARY To determine if an Antarctic species repairs DNA at rates equivalent to warmer water equivalents, we examined repair of UV-damaged DNA in echinoid embryos and larvae. DNA repair by photoreactivation was compared in three species Sterechinus neumayeri (Antarctica), Evechinus chloroticus (New Zealand) and Diadema setosum (Tropical Australia) spanning a latitudinal gradient from polar (77.86°S) to tropical (19.25°S) environments. We compared rates of photoreactivation as a function of ambient and experimental temperature in all three species, and rates of photoreactivation as a function of embryonic developmental stage in Sterechinus. DNA damage was quantified from cyclobutane pyrimidine dimer (CPD) concentrations and rates of abnormal embryonic development. This study established that in the three species and in three developmental stages of Sterechinus, photoreactivation was the primary means of removing CPDs, was effective in repairing all CPDs in less than 24 h, and promoted significantly higher rates of normal development in UV-exposed embryos. CPD photorepair rate constant (k) in echinoid embryos ranged from 0.33 to 1.25 h-1, equating to a time to 50% repair of between 0.6 and 2.1 h and time to 90%repair between 3.6 and 13.6 h. We observed that experimental temperature influenced photoreactivation rate. In Diadema plutei, the photoreactivation rate constant increased from k=0.58 h-1 to 1.25 h-1, with a Q10=2.15 between 22°C and 32°C. When compared among the three species across experimental temperatures (-1.9 to 32°C), photoreactivation rates vary with a Q10=1.39. Photoreactivation rates were examined in three developmental stages of Sterechinus embryos, and while not significantly different, repair rates tended to be higher in the younger blastula and gastrula stages compared with later stage embryos. We concluded that photoreactivation is active in the Antarctic Sterechinus, but at a significantly slower (non-temperature compensated) rate. The low level of temperature compensation in photoreactivation may be one explanation for the relatively high sensitivity of Antarctic embryos to UV-R in comparison with non-Antarctic equivalents.

Cold tolerance of an Antarctic nematode that survives intracellular freezing: comparisons with other nematode species

Panagrolaimus davidi is an Antarctic nematode with very high levels of cold tolerance. Its survival was compared with that of some other nematodes (P. rigidus, Rhabditophanes sp., Steinernema carpocapsae, Panagrellus redivivus and Ditylenchus dipsaci) in both unacclimated samples and those acclimated at 5°C. Levels of recrystallization inhibition in homogenates were also compared, using the splat-cooling assay. The survival of P. davidi after the freezing of samples was notably higher than that of the other species tested, suggesting that its survival ability is atypical compared to other nematodes. In general, acclimation improved survival. Levels of recrystallization inhibition were not associated with survival but such a relationship may exist for those species that are freezing tolerant.

Phylogenetic analysis of three lipocalin-like proteins present in the milk of Trichosurus vulpecula (Phalangeridae, Marsupialia).

Abstract. Three proteins have been identified in the milk of the common brush tail possum, Trichosurus vulpecula that from sequence analysis are members of the lipocalin family. They include β-lactoglobulin, which appears to have two forms; a homologue to the late-lactation protein found in tammar, Macropus eugenii; milk; and a novel protein termed trichosurin. Whereas β-lactoglobulin and trichosurin are both expressed throughout lactation, the late-lactation protein is not detected in samples taken before days 100–110 of lactation. The cDNAs encoding each of these proteins have been isolated from cDNA libraries prepared using possum mammary mRNA and sequenced. Phylogenetic analysis showed that the T. vulpeculaβ-lactoglobulin, along with two other macropod β-lactoglobulins, forms a subclass of β-lactoglobulins distinct from those for eutherian mammals; both marsupial late-lactation proteins appear to have similarities to a family of odorant-binding proteins, whereas trichosurin has similarities to the major urinary proteins of rodents.

Lysozyme and alpha-lactalbumin from the milk of a marsupial, the common brush-tailed possum (Trichosurus vulpecula).

Lysozyme and alpha-lactalbumin have been identified using N-terminal sequence analysis of whey proteins from the common brush-tailed possum, Trichosurus vulpecula after separation by two-dimensional denaturing electrophoresis. Both proteins were purified from pooled possum milk using ion exchange chromatography and gave mass values of 14,896 and 13,985 Da respectively by MALDI-TOF mass spectrometry. Clones containing the full coding sequences of the genes for both proteins were isolated from a possum mammary cDNA library and the DNA sequence of the coding region determined. The inferred protein sequences were used in phylogenetic analysis of both protein classes. These showed that the T. vulpecula alpha-lactalbumin, along with other marsupial alpha-lactalbumins, formed a family distinct from the eutherian alpha-lactalbumins and the alpha-lactalbumin of a monotreme, the platypus, consistent with the separate evolution of the marsupials. By contrast the T. vulpecula lysozyme was shown to be similar to the ruminant stomach lysozymes and primate lysozymes and quite distinct from the Ca2+-binding lysozymes found in the milk of the echidna and horse.

Ice-active proteins and cryoprotectants from the New Zealand alpine cockroach, Celatoblatta quinquemaculata.

Celatoblatta quinquemaculata is moderately freezing tolerant. We have investigated low and high molecular weight compounds that may be associated with its survival. Glycerol and trehalose were identified as potential cryoprotectants, with trehalose at the higher concentration. Trehalose was at its highest concentration in late autumn, during the periods sampled. Water contents declined with time and were significantly lower in late autumn than in late summer. No thermal hysteresis activity was detected in haemolymph or in extracts of the head, muscles and the fat body. Extracts of the Malpighian tubules showed an hexagonal crystal growth form, as did those of the gut tissue and gut contents. The gut tissue had high levels of thermal hysteresis (approximately 2 degrees C) and the gut contents somewhat lower levels (approximately 0.6 degrees C). Recrystallization inhibition activity mirrored that of thermal hysteresis, with activity absent in the haemolymph or fat body cells but present in the gut tissues and contents. Activity was reduced by heating and was associated with a molecule >14kDa in size. These findings suggest an antifreeze protein is involved. In fed animals, ice nucleation is likely to start in the gut. Gut cells have a much greater resistance to freezing than do fat body or Malpighian tubule cells. The antifreeze protein may enable this tissue to survive freezing stress by inhibiting recrystallization.

Multilocus analyses of an Antarctic fish species flock (Teleostei, Notothenioidei, Trematominae): Phylogenetic approach and test of the early-radiation event

Clades that have undergone episodes of rapid cladogenesis are challenging from a phylogenetic point of view. They are generally characterised by short or missing internal branches in phylogenetic trees and by conflicting topologies among individual gene trees. This may be the case of the subfamily Trematominae, a group of marine teleosts of coastal Antarctic waters, which is considered to have passed through a period of rapid diversification. Despite much phylogenetic attention, the relationships among Trematominae species remain unclear. In contrast to previous studies that were mostly based on concatenated datasets of mitochondrial and/or single nuclear loci, we applied various single-locus and multilocus phylogenetic approaches to sequences from 11 loci (eight nuclear) and we also used several methods to assess the hypothesis of a radiation event in Trematominae evolution. Diversification rate analyses support the hypothesis of a period of rapid diversification during Trematominae history and only a few nodes in the hypothetical species tree were consistently resolved with various phylogenetic methods. We detected significant discrepancies among trees from individual genes of these species, most probably resulting from incomplete lineage sorting, suggesting that concatenation of loci is not the most appropriate way to investigate Trematominae species interrelationships. These data also provide information about the possible effects of historic climate changes on the diversification rate of this group of fish.

Characterization of a family of ice-active proteins from the Ryegrass, Lolium perenne☆

Five genes coding for ice-active proteins were identified from an expressed sequence tag database of Lolium perenne cDNA libraries. Each of the five genes were characterized by the presence of an N-terminal signal peptide, a region enriched in hydrophilic amino acids and a leucine-rich region in four of the five genes that is homologous with the receptor domain of receptor-like protein kinases of plants. The C-terminal region of all five genes contains sequence homologous with Lolium and Triticum ice-active proteins. Of the four ice-active proteins (IAP1, IAP2, IAP3 and IAP5) cloned, three could be expressed in Escherichia coli and recovered in a functional form in order to study their ice activity. All three ice-active proteins had recrystallization inhibition activity but showed no detectable antifreeze or ice nucleation activity at the concentration tested. IAP2 and IAP5 formed distinct hexagonal-shaped crystals in the nanolitre osmometer as compared to the weakly hexagonal crystals produced by IAP3.