Osmolyte-induced changes in protein conformational equilibria

Saunders, Aleister J.; Davis-Searles, Paula R.; Allen, Devon L.; Pielak, Gary J.; Erie, Dorothy A.

doi:10.1002/(sici)1097-0282(20000405)53:4<293::aid-bip2>3.0.co;2-t

Cited by 159 publications

(163 citation statements)

References 73 publications

Supporting

Mentioning

145

Contrasting

Unclassified

Order By: Relevance

“…Ubiquitin, like CI2 [20,29], FKBP12 [30,60], lysozyme [22,61,62], ferri-cytochrome C [22,21] and several other proteins increases stability in solutions containing both small osmolytes and larger macromolecules. The equilibrium free energy of unfolding for ubiquitin WT* and the three mutants I13A, K27A and I61V increased by around 1.3±0.4 kcal/mol in the presence of 200 g/L of glucose, a value consistent with other proteins in solutions containing polyols.…”

Section: Discussionsupporting

confidence: 89%

“…This relationship was expected from previous studies on other proteins [16,21]. Comparing values at fixed densities of solute suggests that the increases in WT* stability in glucose and sucrose are within error, as they were when comparing glucose to dextran at the same glucose concentration (Table 2).…”

Section: Equilibrium Unfolding In Sucrosementioning

confidence: 99%

“…In vitro experiments with carbohydrate-based macromolecules and osmolytes suggest that most proteins increase in stability with cosolute concentration in a linear, additive fashion [16,17], with typical increases in stability of around 1-2 kcal/mol in 200-300 g/L of cosolute [16,[18][19][20][21][22]. These stability changes approximate those made by excluded volume theory, which estimates the change 2 in stability of a protein on the basis of the difference between the compactness of the native and denatured states [23].…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Destabilised mutants of ubiquitin gain equal stability in crowded solutions

Roberts¹,

Jackson²

2007

Biophysical Chemistry

View full text Add to dashboard Cite

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT AbstractThis paper investigates the thermodynamic and kinetic response of WT* ubiquitin (F45W) and three mutants to high concentrations of glucose, sucrose and dextran under physiological temperature and pH. WT* ubiquitin was stabilised by the same amount when comparing each cosolute on a weight to volume ratio, with cosolute effects largely independent of denaturant concentration. The energy difference between the mutants and WT* ubiquitin also remained the same in high concentrations of cosolute. An apparent decrease in transition-state surface burial in the presence of the cosolutes was attributed to increased compaction of the denatured state, and not to the Hammond effect. Together, these results suggest higher thermodynamic stabilities and folding rates for proteins in vivo compared to in vitro, in addition to more compact denatured states. Because the effects of mutation are the same in dilute solution and crowded conditions used to mimic the cellular environment, there is validity in using measurements of mutant stabilities made in dilute solutions to inform on how the mutations may affect stability in vivo.

show abstract

Section: Discussionsupporting

confidence: 89%

Section: Equilibrium Unfolding In Sucrosementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Destabilised mutants of ubiquitin gain equal stability in crowded solutions

Roberts¹,

Jackson²

2007

Biophysical Chemistry

View full text Add to dashboard Cite

show abstract

“…However, the slopes for the glycine betaine, Me 2 SO, and sucrose data are higher, ranging from 40 to 60. Variable slopes are common in osmolality studies, but their origin is not clear (37,38,(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54). This issue will be broached under the "Discussion.…”

Section: Role Of Water In K Cat /K M(dhf)supporting

confidence: 81%

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

Chopra

Dooling

Horner

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

R67 dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor. This enzyme is a homotetramer possessing 222 symmetry, and a single active site pore traverses the length of the protein. A promiscuous binding surface can accommodate either DHF or NADPH, thus two nonproductive complexes can form (2NADPH or 2DHF) as well as a productive complex (NADPH⅐DHF). The role of water in binding was monitored using a number of different osmolytes. From isothermal titration calorimetry (ITC) studies, binding of NADPH is accompanied by the net release of 38 water molecules. In contrast, from both steady state kinetics and ITC studies, binding of DHF is accompanied by the net uptake of water. Although different osmolytes have similar effects on NADPH binding, variable results are observed when DHF binding is probed. Sensitivity to water activity can also be probed by an in vivo selection using the antibacterial drug, trimethoprim, where the water content of the media is decreased by increasing concentrations of sorbitol. The ability of wild type and mutant clones of R67 DHFR to allow host Escherichia coli to grow in the presence of trimethoprim plus added sorbitol parallels the catalytic efficiency of the DHFR clones, indicating water content strongly correlates with the in vivo function of R67 DHFR.R67 dihydrofolate reductase, a type II DHFR, 2 catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate. The gene for this enzyme is carried by an R-plasmid, and its presence confers resistance to the antibiotic drug, trimethoprim (TMP). Trimethoprim is a competitive inhibitor of chromosomal DHFR with a picomolar K i (1). Although R67 DHFR is not a good catalyst, it is not inhibited by TMP very well, and thus it allows cell growth in the presence of this antibacterial drug (2, 3). R67 DHFR shares no homology in sequence or structure with chromosomal DHFR and has been proposed to be a good model of a primitive enzyme (4).R67 DHFR is a homotetramer with a single active site pore; the overall structure possesses 222 symmetry as seen in Fig. 1 (5). The symmetry of the active site results in overlapping binding sites for substrate, DHF, and cofactor, NADPH. This can be observed as R67 DHFR binds a total of two ligands as follows: either two NADPH molecules or two folate/DHF molecules or one NADPH plus one folate/DHF molecule (6). The first two complexes are dead-end (binary) complexes, whereas the third is the productive ternary complex. Because of the 222 symmetry, binding to either ligand is unlikely to be optimal.The active site pore of R67 DHFR is unusual in its hourglass shape as well as its large size (2938 Å 3 for apoR67 DHFR with hydrogens added, calculated by CASTp (4, 7)). Because of this large volume, DHF and NADPH cannot occupy all the space in the pore and must use water to mediate some contacts with the protein.How do substrate and cofactor bind to R67 DHFR? From NMR, crystallography, and docking studies, the pteridine ring of DHF/fo...

show abstract

“…Isso significa que o osmólito estabiliza o estado nativo de uma proteína em relação ao estado desnaturado 32,[64][65][66][67][68] . É possível que a estabilização osmofóbica de uma proteína esteja associada ou compreenda a ação conjunta de outros fatores, como a exclusão espacial do osmólito e alterações na constante dielétrica e na viscosidade da solução 36,37,50,[72][73][74][75] . O tratamento dado pela aplicação das funções de Wyman à interação de proteínas com osmólitos apresenta uma validade muito mais ampla e pode justificar uma gama muito variada de fenô-menos aparentemente dissociados, como o equilíbrio N-D e o equilíbrio R-T 21 .…”

Section: Equilíbrio De Estabilização De Proteínas Em Soluções Aquosasunclassified

Efeito da composição do solvente sobre a estabilidade de proteínas em soluções aquosas

Fonseca

Corrêa²,

Garrote-Filho³

et al. 2006

Quím. Nova

View full text Add to dashboard Cite

Recebido em 14/3/05; aceito em 21/7/05; publicado na web em 8/2/06 EFFECTS OF THE SOLVENT COMPOSITION ON THE STABILITY OF PROTEINS IN AQUEOUS SOLUTIONS. A protein presents a native (N) macro state, which is functionally active, in equilibrium with the denatured (D) macro state, which is devoid of biological activity. An ensemble of microstates forms each macrostate. The denatured state comprises a greater ensemble of microstates than the native macrostate. The N-D equilibrium can be affected by several factors, that comprise the purity of the water, temperature, pH and solute concentration. This work discusses the influence of osmolytes and chaotropics on the N-D equilibrium in aqueous solutions.Keywords: chaotropics; protein stability; osmolytes. INTRODUÇÃOAs proteínas são fundamentais para os seres vivos e podem desempenhar diversas funções, desde a organização estrutural de uma célula até as organizações anatômicas de um organismo vivo, além de atuarem como enzimas, que catalisam as mais diversas reações metabólicas indispensáveis à vida.Uma proteína pode estar no estado nativo (N), em que apresenta a função para a qual foi produzida, ou no estado desnaturado (D), em que não apresenta uma função específica, embora ainda seja uma fonte de aminoácidos, que podem ser usados no catabolismo energético e em vários processos de biossíntese.Uma proteína pode apresentar inúmeras conformações. Cada uma delas caracteriza um micro-estado. O conjunto ("ensemble") desses micro-estados compõe o macro-estado da proteína. O macroestado N é composto por uma quantidade pequena de micro-estados, uma vez que a estrutura da proteína não deve variar muito, para não comprometer sua função. Mas o macro-estado D pode apresentar um número muito elevado de micro-estados 1,2 . Os estados N e D de uma proteína estão em equilíbrio N Donde predomina o estado que apresenta menor conteúdo de energia livre e, portanto, maior estabilidade 1-4 . Em soluções essencialmente aquosas, o estado N tem maior estabilidade, o que é fundamental para preservar a função da proteína. Vários fatores, como temperatura, pH e concentração de solutos, podem afetar o equilíbrio N-D, promovendo estabilização (do estado nativo) ou desnaturação. A estabilização e a desnaturação de proteínas são temas de grande interesse fisiológico 5 , terapêutico 6,7 e biotecnológico 8,9 , onde o controle da estabilidade de uma proteína representa uma importante estratégia para manutenção de sua atividade biológica. Além disso, atualmente a análise da estabilidade se tornou um instrumento poderoso para caracterização de proteínas [10][11][12] , cada vez mais utilizado na literatura com este propósito [13][14][15][16] . A estabilização e desnaturação de proteínas têm sido revistas em trabalhos clássi-cos, que mostram a natureza complexa desses processos [17][18][19][20][21][22] . O presente trabalho procura se inserir neste contexto, fazendo uma revisão analítica sobre a natureza termodinâmica do efeito da água pura, de caotrópicos e osmólitos sobre a estabilidade de proteínas em sol...

show abstract

Osmolyte-induced changes in protein conformational equilibria

Cited by 159 publications

References 73 publications

Destabilised mutants of ubiquitin gain equal stability in crowded solutions

Destabilised mutants of ubiquitin gain equal stability in crowded solutions

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

Efeito da composição do solvente sobre a estabilidade de proteínas em soluções aquosas

Contact Info

Product

Resources

About