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hyperthermpohiles

hyperthermpohiles

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Protein Stability at High Pressure and Temperature 101
Adaptation of Proteins fromHyperthermophiles to HighPressure and High Temperature
*For correspondence. Email robb@umbi.umd.edu;Tel. 410-234 8870; Fax. 410-234 8896.
J. Molec. Microbiol. Biotechnol. (1999) 1(1): 101-105.
 © 1999 Horizon Scientific Press
 
JMMB Symposium
Frank T. Robb
1
*, and Douglas S. Clark
2
1
Center of Marine Biotechnology, University of MarylandBiotechnology Institute, Baltimore, MD 21202, USA
2
Department of Chemical Engineering, University ofCalifornia, Berkeley, USA
AbstractFurther clarification of the adaptations permitting thepersistence of life at temperatures above 100
°
Cdepends in part on the analysis of adaptivemechanisms at the protein level. The hypertherm-ophiles include both Bacteria and Archaea, althoughthe majority of isolates growing at or above 100
°
C areArchaea. Newly described adaptive features ofhyperthermophiles include proteins whose structuralintegrity persists at temperatures up to 200
°
C, andunder elevated hydrostatic pressure, which in somecases adds significant increments of stability.Introduction
It is now very well established that microbial growth canoccur at temperatures well above 100
°
C. The recentlydescribed microorganism with the highest recorded growthtemperature of 113.5
°
C, is the chemolithoautotrophicarchaeon
Pyrolobus fumarii 
(Blochl
et al.
, 1997).Hyperthermophiles, which are defined as organisms withmaximal growth temperatures above 90
°
C (Adams, 1994),are widely distributed in hydrothermal environments interrestrial as well marine and abyssal vent systems. Thegroup includes 21 archaeal genera, and two bacterialgenera with diverse growth physiology including bothheterotrophs and chemoautotrophs (Stetter, 1996; Robb
et al.
, 1995). The archaeal hyperthermophiles haveexceptionally high growth temperature limits, unique,molecular characteristics, such as ether-linked lipids andeukaryotic transcription and translation factors (Robb
et al.
1995), and unusual enzyme chemistry, such as thepresence of multiple tungsten-containing enzymes (Kletzinand Adams, 1996). The unusual adaptive responses of thehyperthermophiles include heat shock proteins of the hsp60family (Trent, 1996; Trent
et al.
, 1997; Kagawa
et al.
, 1995)which are inducible by supraoptimal temperatures, andreverse gyrase which installs positive supercoils in DNA(Guipaud
et al.
, 1997; Borges
et al.
, 1997).The ongoing release of genomic sequence data fromhyperthermophiles will continue to accelerate the discoveryof thermostable proteins, however understanding thefunctional adaptations of these proteins requires theapplication of novel methods of analysis. For example, twokey enzymes in glycolysis, phosphofructokinase andhexokinase, remained undiscovered in extracts of thehyperthermophile
Pyrococcus furiosus 
because it wasanticipated that they would require ATP. It transpires thatenzymes are active and highly expressed, but require ADP(Kengen
et al.
, 1995), presumably due to the higher stabilityof ADP compared with ATP. This review focuses onmechanisms of stabilization of enzymes with novel catalyticspecificities, unusual thermal stability, and the ability towithstand, and in many cases to gain enhancedthermostability under elevated hydrostatic pressure. Thelatter is an unusual and intriguing feature of many proteinsfrom thermophiles and recent work suggests that thestructural stability of proteins in the 100-130
°
C range isachieved by quite generic adaptive mechanisms. We willreview the current field of protein stability at very hightemperatures and discuss recent findings on the theoreticalbasis for pressure stabilization of proteins.
Mechanisms of Hyperstability
The known strategies for maintaining stability at hightemperatures are summarized in Table 1. Five classes ofadaptations are discussed below.
1. Amino Acid Composition
Although many formulae for compositional bias inhyperstable proteins have been suggested, there appearto be few answers to the quest for a thermoadapted aminoacid composition. On the contrary, hyperstable proteins,like their counterparts in mesophiles, are composed of thesame 20 amino acid set found in mesophiles. These aminoacids undergo accelerated covalent modification at the hightemperatures, pressures and extremes of pH that manythermophiles and hyperthermophiles can withstand (Hensel
et al.
, 1992). At temperatures beyond 100
°
C, the stabilityof common amino acids declines in the following order:(Val,Leu)>Ile>Tyr>Lys>His>Met>Thr>Ser> Trp>(Asp, Glu,Arg, Cys) (Jaenicke 1991; Jaenicke 1996a; Jaenicke andBohm, 1998). Since many of the hyperthermophiles arerelatively slow growing, the half lives of some of the morelabile amino acids such as Cys, Arg, Gln, Asn areconsiderably shorter than the generation times of theorganisms (Bernhardt
et al.
, 1984). Common types ofchemical alterations include deamidation, ß-oxidation,Maillard reactions, hydrolysis, and disulfide interchange,etc (Jaenicke and Bohm, 1998). Many of the amino acidsare more stable upon internalization in the hydrophobiccore of the proteins than in free solution (Hensel
et al.
,1992), however the frequencies of occurrence of Cys, Asn,Gln and Asp is significantly lowered in the hyperstablevariants of some common proteins. The genomicsequences that are determined or in progress will continueto make available a flood of deduced protein sequences,
 
102 Robb and Clark
which will lead to additional insights in the area ofcompositional bias. Compatible solutes, some of whichhave recently been found to provide genericthermoprotection to proteins, may be acting byincrementally slowing amino acid modification reactionsat high temperatures (Hensel
et al.
, 1992).
2. Hydrophobic Packing
An extensive, efficiently packed hydrophobic core is acommon feature of stable globular proteins, although itappears to be more dominant in the lesser hyper-thermophiles than in those growing at or above 100
°
C(Russell
et al.
, 1995; Macedo-Ribeiro, 1997; Pfeil
et al.
,1997; Jaenicke, 1996b).At present, the effect of temperature on hydrophobicinteractions is still a subject of debate in the literature. Thestrength of the hydrophobic interaction is most commonlyrepresented by
°  
tr 
, the Gibbs free energy of transfer ofhydrocarbons from a pure hydrocarbon liquid to water(Schellman, 1997). An alternative is to define the strengthof the hydrophobic interaction by
°  
tr 
 /T 
, which isproportional to the natural log of the equilibrium constant(Schellman, 1997). The temperature dependence of thestrength of the hydrophobic interaction depends on whichdefinition is used. In particular, plots of
°  
tr 
vs. T for modelhydrocarbons exhibit a maximum at around 140
°
C(Privalov and Gill, 1988), whereas curves of
°  
tr 
 /T 
vs. Thave a maximum at around 20
°
C (Privalov and Gill, 1988).Thus, it is still unclear whether hydrophobic interactions inproteins become stronger or weaker at temperatures aboveroom temperature, and how important they are to thestability of thermophilic proteins at high temperatures.Interestingly, some of the same constraints appear tooperate in limiting the stability of proteins at lowtemperatures, between –10 and 20
°
C (Privalov and Gill,1988). There is also evidence that hyperstable glutamatedehydrogenase requires high temperatures for assembly,suggesting that specific hydrophobic states are critical tothe molecular engagement of subunits, and that partlyassembled multimeric proteins may be “frozen” inmetastable intermediates. This system may provideinsights into the nature of the folding and assemblypathways of hyperstable proteins (DiRuggiero and Robb,1995; 1996).
3. Ionic Networks
Extensive networks of acidic and basic side chains on thesurface of subunits and domains interact to formcooperatively bound assemblages (Russell and Taylor,1995; Robb and Maeder, 1998; Rice
et al.
, 1996; Britton
et al.
, 1995). Ionic interactions by nature act over much longerranges than hydrophobic interactions, and are relativelyimmune to alterations in the structure of water which iscompromised at elevated temperatures. Consequently,widespread networks of ionic interactions are observed inthe proteins of the more extreme hyperthermophilescompared with homologous proteins in the thermophiles(T
opt
for growth between 50 and 75
°
C) or in mesophiles(Osterdorp
et al.
, 1996; Britton
et al.
, 1995). The ionicnetworks of
P.furiosus 
glutamate dehydrogenasedemonstrate extensive clustering of spatially alternatingpositive and negative charges, which have a componentof hydrogen bonding. Recreating a network in the lessstable glutamate dehydrogenase from
Thermococcus litoralis 
resulted in elevated thermostability without anypenalty in catalytic activity (Vetriani
et al.
, 1998).
4. Cooperative Association
In proteins that form oligomers or bind to larger substratesthan themselves, dissociation is thought to precede theirreversible unraveling of monomers. The loss of integrityof the protein molecule is dependent on the unfolding ofthe monomeric protein into the denatured form. It followsthat strong intermolecular associations can forestall thisprocess. Consequently, many proteins that are monomericin mesophiles are found to form oligomers in extremethermophiles or hyperthermophiles. For example, thechorismate mutase from the hyperthermophile,
M. jannaschii 
, appears to have developed a dimeric quaternaryorganization as an adaptation to stability (MacBeath
et al.
,1998), and the compact beta-alpha TIM barrel ofphosphoribosyl anthranilate isomerase, which ismonomeric in enteric bacteria, is dimeric in
T. maritima 
(Hennig
et al.
, 1997),
5. Pinning the Loose Ends
It is thought that many N- and C-termini are immobilized inhyperstable proteins, and are therefore not free to fray andinitiate unraveling. In the case of phosphoribosylanthranilate isomerase from
T.maritima 
(Hennig
et al.
,
Table 1. Synopsis of Mechanisms of Extreme Thermostability in Proteins from HyperthermophilesOrganism (Topt)Thermostability featureProteinReference
Pyrococcus furiosus 
(100
°
C)Mg
++
requirement KCl (1M)Acetyl-CoA synthetase (ADP-forming)Glasemacher
et al.
, 1997
P. furiosus 
(100
°
C)Salt bridgesGlutamate dehydrogenaseKlump
et al.
, 1992; Vetriani
et al.
, 1998
Sulfolobus acidocaldarius 
(85
°
C)Lysine monomethylationSac7dMcCrary
et al.
, 1996NOT salt bridges
S. acidocaldariu
(85
°
C)The data conforms to noneAdenylate kinaseSchafer
et al.
, 1996of the sequence basedPyrophosphataseconventions proposed by othersSuperoxide dismutase
Methanothermus fervidus 
(65
°
C)Hydrophobic proline N capsHistone rHMfBStarich
et al.
, 1996Interhelical hydrogen bondsShort N- and C-termini
Aquifex pyrophilus 
(98
°
C)Salt bridgesSuperoxide dismutase (SOD)Lim
et al.
, 1997Hydrophobic packingPolymeric tetramer
Thermotoga maritima 
(77
°
C)HomodimerPhosphoribosyl anthranilate isomeraseHennig
et al.
, 1997
T. maritima 
(77
°
C)Replace loop with helixPhosphoribosyl anthranilate isomeraseHennig
et al.
, 1997Homo-dimerhydrophobic packingN- and C- termini immobilizedSalt bridges
 
Protein Stability at High Pressure and Temperature 103
1997), not only are the termini buried in hydrophobicpockets, but a disordered loop found in the homologousprotein from
E. coli 
is replaced with an alpha helix, therebyinhibiting the nucleation of melting. The C-termini of thedimeric citrate synthases of hyperthermophiles has anintertwined structure which contributes stability by lockingthe dimer together as well as securing the carboxyl groupsby ionic interaction, preventing disengagement of the ends(Russell
et al.
, 1997). The protein with the highesttemperature stability on record,
P. furiosus 
ferredoxin, whichretains structure at temperatures up to 200
°
C (Hiller
et al.
, 1997), features an ion-pair at the amino terminus(Cavagnero
et al.
, 1998). Loose ends must be handledsomewhat differently in the multiple proteins fromhyperthermophiles that have inteins. For example, theribonucleotide reductase from
Pyrococcus furiosus 
hasbeen shown to have two inteins (Riera
et al.
, 1997), andthe mechanisms of intein excision have been shown toproceed at optimal growth temperatures (Perler
et al.
,1997), implying that there is an end-stabilizing mechanismfor these protein splicing reactions.
Pressure Stabilization
Many hyperthermophiles isolated from deep seahydrothermal vents are either indifferent to the effects ofpressure on growth at high temperature, or else they arebarophilic in terms of maximal growth rate and the uppertemperature limits of growth (Miller
et al.
, 1988; Nelson
et al.
, 1992; Pledger
et al.
, 1994; Marteinsson
et al.
, 1997).Although many studies of pressure effects on enzymestability have appeared since the early 1950’s, in nearlyall cases the experiments were performed with proteinsfrom mesophilic sources at temperatures below 60
°
C, suchas lysozyme (Samarasighe
et al.
, 1992). Moreover, muchof this early work focused on enzyme denaturation at veryhigh pressures (> 300 MPa) (Jaenicke, 1991; Weber andDrickamer, 1983). More recent work, however, has shownthat moderate pressures (100 MPa) below those normallyneeded for pressure-induced denaturation can in factstabilize proteins against thermoinactivation (Hei and Clark,1994; Michels and Clark, 1997; Michels
et al 
., 1996;Mozhaev
et al 
., 1996). Particularly large effects have beenobserved for thermophilic enzymes at very hightemperatures. This behavior has important implications forthe adaptation of thermophilic proteins
in extremis 
, asdiscussed more fully below.In the first report of thermophilic enzyme stabilizationby pressure, Hei and Clark (1994) examined the effect ofpressure on the thermal stability of four partially purifiedhydrogenases from methanogens of the genus
Methanococcus.
Only one of these organisms,
M. jannaschii 
, was isolated from a deep-sea habitat: a deep-sea hydrothermal vent at a depth of 2500 m (Jones
et al.
,1983). Notably, hydrogenases from the extremethermophiles
M. jannaschii 
and
M. igneus 
weresubstantially stabilized by pressure whereas hydrogenasesfrom
M. thermolithotrophicus 
(a moderate thermophile) and
M. maripaludis 
(a mesophile) were destabilized bypressure. These results were the first demonstration ofpressure stabilization for thermophilic enzymes, andshowed that the effect is not unique to enzymes isolatedfrom high-pressure environments. The work of Summit
et al.
(1998) confirmed that the DNA polymerases from non-barophilic thermophiles could be stabilized by pressure.Furthermore, the hydrogenase studies, in combination withstudies on the effects of pressure on several homologousglyceraldehyde-3-phosphate dehydrogenases frommesophilic and thermophilic sources and a rubredoxin from
P. furiosus 
, implicated hydrophobic interactions as animportant factor in the stabilization of thermophilic enzymesby pressure.In follow-up work to the studies of Hei and Clark (1994),Michels and Clark (1997) isolated a proteolytic enzymefrom
M. jannaschii 
and found that the enzyme was bothactivated and stabilized by pressure. Even at moderatepressures, the thermal half-life of the enzyme wasexceptionally high; for example, t
1/2
= 45 min at 116
°
C,and 7 min at 125
°
C (at ~10 atm pressure). Moreover, thethermostability of the
M. jannaschii 
protease increased withpressure in a physiologically-relevant range. For example,by raising the pressure to 500 atm, the half-life of theenzyme was increased 2.7-fold at 125
°
C. The pronouncedeffect of pressure on the
M. jannaschii 
protease stabilitywas unusual for proteolytic enzymes: when 500 atm wasapplied to trypsin,
α
-chymotrypsin, and subtilisin Carlsberg,half-lives at the enzyme’s reported melting temperatureincreased by 40%, 30% and 30%, respectively, comparedto 170% for
M. jannaschii 
protease at 125
°
C (Michels andClark, 1997).The barophilic behavior of the
M. jannaschii 
protease(and of the corresponding hydrogenase; Miller
et al.
, 1989)is especially noteworthy in view of the unusual barophilyexhibited by
M. jannaschii 
in high-pressure growthexperiments (Miller
et al.
, 1988). Whether such pressure-activation and stabilization of key enzymes is responsiblefor the barophilic growth of
M. jannaschii 
remains to beseen; however, the similar effects of pressure on growthand enzyme activity suggests that these phenomena areinterrelated and motivates further pressure studies ofenzymes from
M. jannaschii 
and other deep-seathermophiles.In more recent work, Sun
et al.
(1999) examined thethermostability and pressure-induced thermostabilizationof glutamate dehydrogenase (GDH) from thehyperthermophilic archaeon
Pyrococcus furiosus 
(
Pf 
) attemperatures up to 109
°
C (
Pf 
GDH, a hexamer composedof six identical subunits, has a melting temperature fordenaturation of 113
°
C). The native GDH from
Pf 
was sub-stantially stabilized by 500 atm, up to 18-fold at 109
°
C. Bycomparison, a recombinant GDH mutant containing anextra tetrapeptide at the C-terminus was stabilized to aneven greater degree, by 28-fold at 105
°
C. Although thepresence of the tetrapeptide destabilized the enzymemarkedly, the destabilizing effect was largely reversed bypressure. The remarkable degree of pressure stabilization,especially of the recombinant GDH mutant, could not beattributed to hydrophobic interactions alone. However,further insights into the possible mechanism(s) of pressurestabilization were provided by inactivation experiments inthe presence of glycerol. Specifically, stabilization was alsoachieved by adding glycerol, albeit to a lesser extent thaneffected by pressure, suggesting that compression and/orrigidification of the protein’s structure played a role inpressure-induced thermostabilization.Of the primary intramolecular interactions responsiblefor maintaining native protein structure, only hydrophobicinteractions are expected to be stabilized by elevated

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