Characterization of the restricted rotation of the dimethyl groups in chemically N-terminal C13-labeled antifreeze glycoproteins: A temperature-dependent study in water to ice through the supercooled state
Abstract:Site-specific chemical modification, especially with isotopically enriched groups, allows one to study the structure and dynamics of proteins for which uniform enrichment is difficult. When the N-terminal alanine in antifreeze glycoprotein (AFGP) is replaced with an N,N-dimethyl alanine the methyl groups show signatures of slow rotation about the C-N bond. In order to separate the local dynamics of the N-terminus from the overall protein dynamics, we present a complete characterization of this dynamics. Temper… Show more
“…Because the area of the 1 δ > 5 ppm signals of Figure , A w , gauges the amount of liquid water in frozen solutions, , and A w vanishes below ∼250 K (Figure ), water seems to freeze completely at the point at which Q H becomes independent of temperature. On the other hand, the line widths of methyl proton resonances, which gradually increase at lower temperatures (Figure ), abruptly broaden below ∼230 K (i.e., about 20 K below the disappearance of mobile water) as an indication that internal rotations of methyl groups become too slow to effectively average local magnetic anisotropy, , at temperatures that are commensurate with the glass transition of bulk supercooled D 2 O, T g = 233 K. , DSC thermograms of frozen PA solutions, which display a single endotherm at the onset of ice melting (Supporting Information), further confirm that PA aqueous solutions freeze into ice and a fluid solution that eventually vitrifies at ∼230 K. 5 Integral intensity of the 1 δ ≳ 5 ppm water signals as function of temperature. (▵) in 0.10 M PA. (○) in 0.16 M PA/0.13 M HCl. 6 (○) 1 δ: ≈ 2.4 ppm and (▵) ∼1.5 ppm signal line widths at half-height as function of temperature in frozen 0.1 M PA in D 2 O.…”
About 3.5 ( 0.3 water molecules are still involved in the exothermic hydration of 2-oxopropanoic acid (PA) into its monohydrate (2,2-dihydroxypropanoic acid, PAH) in ice at 230 K. This is borne out by thermodynamic analysis of the fact that QH(T) ) [PAH]/[PA] becomes temperature independent below ∼250 K (in chemically and thermally equilibrated frozen 0.1 e [PA]/M e 4.6 solutions in D2O), which requires that the enthalpy of PA hydration (∆HH ∼ -22 kJ mol -1 ) be balanced by a multiple of the enthalpy of ice melting (∆HM ) 6.3 kJ mol -1 ). Considering that: (1) thermograms of frozen PA solutions display a single endotherm, at the onset of ice melting, (2) the sum of the integral intensities of the 1 δPAH and 1 δPA methyl proton NMR resonances is nearly constant while, (3) line widths increase exponentially with decreasing temperature before diverging below ∼230 K, we infer that PA in ice remains cooperatively hydrated within interstitial microfluids until they vitrify.
“…Because the area of the 1 δ > 5 ppm signals of Figure , A w , gauges the amount of liquid water in frozen solutions, , and A w vanishes below ∼250 K (Figure ), water seems to freeze completely at the point at which Q H becomes independent of temperature. On the other hand, the line widths of methyl proton resonances, which gradually increase at lower temperatures (Figure ), abruptly broaden below ∼230 K (i.e., about 20 K below the disappearance of mobile water) as an indication that internal rotations of methyl groups become too slow to effectively average local magnetic anisotropy, , at temperatures that are commensurate with the glass transition of bulk supercooled D 2 O, T g = 233 K. , DSC thermograms of frozen PA solutions, which display a single endotherm at the onset of ice melting (Supporting Information), further confirm that PA aqueous solutions freeze into ice and a fluid solution that eventually vitrifies at ∼230 K. 5 Integral intensity of the 1 δ ≳ 5 ppm water signals as function of temperature. (▵) in 0.10 M PA. (○) in 0.16 M PA/0.13 M HCl. 6 (○) 1 δ: ≈ 2.4 ppm and (▵) ∼1.5 ppm signal line widths at half-height as function of temperature in frozen 0.1 M PA in D 2 O.…”
About 3.5 ( 0.3 water molecules are still involved in the exothermic hydration of 2-oxopropanoic acid (PA) into its monohydrate (2,2-dihydroxypropanoic acid, PAH) in ice at 230 K. This is borne out by thermodynamic analysis of the fact that QH(T) ) [PAH]/[PA] becomes temperature independent below ∼250 K (in chemically and thermally equilibrated frozen 0.1 e [PA]/M e 4.6 solutions in D2O), which requires that the enthalpy of PA hydration (∆HH ∼ -22 kJ mol -1 ) be balanced by a multiple of the enthalpy of ice melting (∆HM ) 6.3 kJ mol -1 ). Considering that: (1) thermograms of frozen PA solutions display a single endotherm, at the onset of ice melting, (2) the sum of the integral intensities of the 1 δPAH and 1 δPA methyl proton NMR resonances is nearly constant while, (3) line widths increase exponentially with decreasing temperature before diverging below ∼230 K, we infer that PA in ice remains cooperatively hydrated within interstitial microfluids until they vitrify.
“…Such a methodology was used to understand the properties of antifreeze proteins. (1,4,34,35,52,53) Likewise, the role of teichoic acid in the low-temperature survival of Gram(+) bacteria is unknown. The peptidoglycan provides a more robust cell envelope for Gram(+) bacteria rather than the weak envelope of lipid bilayers for Gram(-) bacteria.…”
Numerous chemical additives lower the freezing point of water, but life at sub-zero temperatures is sustained by a limited number of biological cryoprotectants. Antifreeze proteins in fish, plants, and insects provide protection to a few degrees below freezing. Microbes have been found to survive at even lower temperatures, although, with a few exceptions, antifreeze proteins are missing. Survival has been attributed to external factors, such as high salt concentration (brine veins) and adhesion to particulates or ice crystal defects. Teichoic acid is a phosphodiester polymer ubiquitous in Gram positive bacteria, composing 50% of the mass of the bacterial cell wall and excreted into the extracellular space of biofilm communities. We have found that when bound to the peptidoglycan cell wall (wall teichoic acid) or as a free molecule (lipoteichoic acid), teichoic acid is surrounded by liquid water at temperatures significantly below freezing. Using solid-state NMR, we are unable to collect 31 P CPMAS spectra for frozen solutions of lipoteichoic acid at temperatures above -60 °C. For wall teichoic acid in D 2 O, signals are not seen above -30 °C. These results can be explained by the presence of liquid water, which permits rapid molecular motion to remove 1 H/ 31 P dipolar coupling. 2 H quadrupole echo NMR spectroscopy reveals that both liquid and solid water are present. We suggest that teichoic acids could provide a shell of liquid water around biofilms and planktonic bacteria, removing the need for brine veins to prevent bacterial freezing.
IntroductionNature relies on chemistry to maintain liquid water for life at subfreezing temperatures. The current paradigms use thermodynamic and/or kinetic arguments for cryosurvival in mammals, plants and insects.(1-3) At the onset of freezing, frogs excrete large amounts of glucose to lower the freezing point of water while preventing the formation of large ice crystals. Fish, plants, and insects inhibit ice formation with antifreeze proteins (AFP) that bind to the growth face of ice crystals. Enthalpic contributions are accompanied by entropic factors from a disorganized chemical system.(4) These factors slow the kinetics of crystallization such that AFPs perform their task at very low concentration, characterized as a non-colligative depression of the freezing point.Little is known of the cryoprotection chemistry for the predominate organisms in cold environments: microbes.(5, 6) Bacteria are found in nearly all low temperature environments and must prevent ice formation to ensure survival. Microbial communities have been discovered in ice,(7) snow,(8) permafrost,(9) and at the polar ice caps.(10, 11) Analysis of core samples show the presence of viable life hundreds of feet below the ice surface. Samples of frozen tundra have yielded various Gram positive, Gram negative, and Archaea species. (9, 12, 13) Here, Gram-negative Cryseobacterium and Gram-positive Enterococci were found to have a symbiotic interaction. The basis for cryosurvival is unknown, but macromolecules an...
“…One possibility is the use of NMR spectroscopy; a methodology was used to understand the properties of antifreeze proteins. (Chao et al 1997; Krishnan et al 2005; Mao and Ba 2006; Scotter et al 2006; Tsvetkova et al 2002; Yeh and Feeney 1996) Additional insight regarding the role of teichoic acids in low temperature biochemistry could be obtained using mutants with teichoic acid deletions. These strains should exhibit increased freeze mortality.…”
Antifreeze proteins in fish, plants, and insects provide protection to a few degrees below freezing. Microbes have been found to survive at even lower temperatures, and with a few exceptions, antifreeze proteins are missing. We show that lipoteichoic acid (LTA), a biopolymer in the cell wall of Gram-positive bacteria, can be added to B. subtilis cultures and increase freeze tolerance. At 1% w/v, LTA enables a 50% survival rate, similar to the results obtained with 1% w/v glycerol as measured with the resazurin cell viability assay. In the absence of added LTA or glycerol, a very small number of B. subtilis cells survive freezing. This suggests that an innate freeze tolerance mechanism exists. While cryoprotection can be provided by extracellular polymeric substances (EPS), our data demonstrate a role for LTA in cryoprotection. Currently, the exact mode of action for LTA cryoprotection is unknown. With a molecular weight of 3-5 kDa, it is unlikely to enter the cell cytoplasm. However, low temperature microscopy data show small ice crystals aligned along channels of liquid water. Our observations suggest that teichoic acids could protect liquid water within biofilms and planktonic bacteria, augmenting the role of brine while also raising the possibility for survival without brine present.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
