Water structure in vitro and within Saccharomyces cerevisiae yeast cells under conditions of heat shock

The OH stretch mode from water and organic hydroxyl groups have strong infrared absorption, the position of the band going to lower frequency with increased H-bonding. This band was used to study water in trehalose and glycerol solutions and in genetically modified yeast cells containing varying amounts of trehalose. Concentration-dependent changes in water structure induced by trehalose and glycerol in solution were detected, consistent with an increase of lower-energy H-bonds and interactions at the expense of higher-energy interactions. This result suggests that these molecules disrupt the water H-bond network in such a way as to strengthen molecule-water interactions while perturbing water-water interactions. The molecule-induced changes in the water H-bond network seen in solution do not translate to observable differences in yeast cells that are trehalose-deficient and trehalose-rich. Although comparison of yeast with low and high trehalose showed no observable effect on intracellular water structure, the structure of water in cells is different from that in bulk water. Cellular water exhibits a larger preference for lower-energy H-bonds or interactions over higher-energy interactions relative to that shown in bulk water. This effect is likely the result of the high concentration of biological molecules present in the cell. The ability of water to interact directly with polar groups on biological molecules may cause the preference seen for lower-energy interactions.     

Protective mechanisms of α,α-trehalose revealed by molecular dynamics simulations

Molecular dynamics simulations were carried out to model aqueous solution with different concentration of α,α-trehalose, one kind of non-reducing sugars possessing outstanding freeze-drying protective effect on biological system. The dihedral angles of the intraglycosidic linkage in trehalose were measured to estimate its structure rigidity. The dynamics and hydrogen bonding properties were studied by calculating the self-diffusion coefficient of trehalose and the distributions and lifetimes of various types of H-bonds in the solution. Through analysing the results as well as comparison with another common sugar sucrose, the freeze-drying protective mechanism of trehalose was explained at molecule level. First, trehalose is able to maintain the local structure around it as a frame due to its relatively rigid conformation. Second, the addition of trehalose restrains the water molecules from rearrangement as a result of low mobility, thus reduces the probability of freezing; trehalose has lower diffusion coefficient than water and bigger thermal diffusivity, which are favourable for vitrification. Third, the formation of H-bonds between trehalose and water and between trehalose molecules is the essence of the protective effect. Trehalose does not work via strengthening the H-bonds formed between water molecules (W–W H-bonds), instead of which it breaks the potential tetrahedral pattern of W–W H-bonds, thus suppresses the tendency of ice formation. It was also found that trehalose realises protective action better at higher concentration as far as this study is concerned.     

Aggregation dependent enhancement of trehalose-6-phosphate synthase activity in Saccharomyces cerevisiae

Trehalose-6-phosphate synthase (TPS) is one of the key subunits of the trehalose synthase complex, responsible for synthesis of trehalose in Saccharomyces cerevisiae. Different laboratories have tried to purify TPS, but have been unable to separate it from the complex. During the present study, active TPS has been isolated from the trehalose synthase complex as a free 59kDa protein. A 158 fold purification was achieved with over 84% recovery of active TPS. N-terminal sequence confirmed the 59kDa protein to be TPS. It was revealed to be a highly hydrophobic protein by amino acid analysis data. Activity of TPS was identified to be governed by association–dissociation of protein components. TPS activity of the isolated enzyme was highly unstable due to dissociation of the protein from the complex. Aggregation of active molecules was also seen to enhance as well as stabilize enzyme activity. This aggregation was concentration dependent and activity was seen to be enhanced by increasing the number of active molecules and fell with dilution. The association of the active complex was also found to be governed by ionic interactions.     

Mannitol production from glycerol by resting cells of Candida magnoliae

Production of mannitol from glycerol by resting cells of Candida magnoliae under aerobic condition was investigated. The resting cells were suspended in aqueous solution of glycerol in Erlenmeyer flasks and incubated on rotary shaker. The samples were analyzed by ion exclusion–HPLC equipped with refractive index and UV detector. The resting cells of C. magnoliae produced mannitol from fructose, sucrose and glycerol but not from glucose. Addition of yeast extract and/or potassium phosphate to the glycerol solution adversely affected its conversion to mannitol. The conversion of glycerol to mannitol was dependent on oxygen availability. Using resting cells, the yield of mannitol was as high as 45%. This is probably the first report of conversion of glycerol to mannitol by osmophilic yeast.     

Hydrogen-bonding interactions in adrenaline–water complexes: DFT and QTAIM studies of structures, properties, and topologies

ωB97XD/6-311++G(d,p) calculations were carried out to investigate the hydrogen-bonding interactions between adrenaline (Ad) and water. Six Ad-H(2)O complexes possessing various types of hydrogen bonds (H-bonds) were characterized in terms of their geometries, energies, vibrational frequencies, and electron-density topology. Natural bond orbital (NBO) and quantum theory of atoms in molecules (QTAIM) analyses were performed to elucidate the nature of the hydrogen-bonding interactions in these complexes. The intramolecular H-bond between the amino and carboxyl oxygen atom of Ad was retained in most of the complexes, and cooperativity between the intra- and intermolecular H-bonds was present in some of the complexes. H-bonds in which hydroxyls of Ad/water acted as proton donors were stronger than other H-bonds. Both hydrogen-bonding interactions and structural deformation play important roles in the relative stabilities of the complexes. The intramolecular H-bond was broken during the formation of the most stable complex, which indicates that Ad tends to break the intramolecular H-bond and form two new intermolecular H-bonds with the first water molecule.     

The induction of trehalose and glycerol in Saccharomyces cerevisiae in response to various stresses

Trehalose and glycerol have been implicated as potential stress protectants that accumulate in yeasts during various stress conditions. We investigated the levels of glycerol and trehalose and the expression profiles of genes involved in their metabolism to determine their involvement in the response of Saccharomyces cerevisiae XQ1 to thermal, sorbitol and ethanol stresses. The results showed that the genes involved in the synthesis and degradation of trehalose and glycerol were stress induced, and that trehalose and glycerol were synthesized simultaneously during the initial stages (a sensitive response period) of diverse stress treatments. Trehalose accumulated markedly under heat treatment, but not under sorbitol or ethanol stress, whereas glycerol accumulated strikingly under sorbitol stress conditions. Interestingly, extracellular trehalose seemed to be involved in protecting cells from damage under unfavorable conditions. Moreover, our results suggest that the stress-activated futile ATP cycles of trehalose and glycerol turnover are of general importance during cellular stress adaptation.     

Conformational studies of sphingolipids by NMR spectroscopy. I. Dihydrosphingomyelin

The conformational features of dihydrosphingomyelin (DHSM), the major phospholipid of human lens membranes, were investigated by 1H and 31P nuclear magnetic resonance spectroscopy. Several postulates emerge from the observed trends: (a) in partially hydrated samples of DHSM in CDCl3 above 13 mM, at which lipid-lipid interactions prevail, the amide proton is mostly involved in intermolecular H-bonds that link neighboring phospholipids through bridging water molecules. In the absence of water, the NH group is involved in an intramolecular H-bond that restricts the mobility of the phosphate group. (b) In the monomeric form of the lipid molecule, the amide proton of the major conformer is bound intramolecularly with one of the anionic and/or ester oxygens of the phosphate group. A minor conformer may also be present in which the NH proton participates in an intramolecular H-bond linking to the OH group of the sphingoid base. (c) Complete hydration leads to an extension of the head group as water molecules bind to the phosphate and NH groups via H-bonds, thus disrupting the intramolecular H-bonds prevalent at low concentrations.     

1H NMR investigation of the solution structure of substrate-free human heme oxygenase: comparison to the cyanide-inhibited, substrate-bound complex

1H NMR was used to investigate the molecular structure, and dynamic properties of soluble, recombinant, substrate-free human heme oxygenase (apohHO) on a comparative basis with similar studies on the substrate complex. Limited but crucial sequence-specific assignments identify five conserved secondary structural elements, and the detection of highly characteristic dipolar or H-bond interactions among these elements together with insignificant chemical shift differences confirm a strongly conserved folding topology of helices C-H relative to that of substrate complexes in either solution or the crystal. The correction of the chemical shifts for paramagnetic and porphyrin ring current influences in the paramagnetic substrate complex reveals that the strength of all but one of the numerous relatively robust H-bonds are conserved in apohHO, and similar ordered water molecules are located near these H-bond donors as observed in the substrate complexes. The unique and significant weakening of the Tyr(58) OH hydrogen bond to the catalytically critical Asp(140) carboxylate in apohHO is suggested to arise from the removal of the axial H-bond acceptor ligand rather than the loss of substrate. The interhelical positions of the conserved strong H-bonds argue for a structural role in maintaining a conserved structure for helices C-H upon loss of substrate. While the structure and H-bond network are largely conserved upon loss of substrate, the variably increased rate of NH lability dictates a significant loss of dynamic stability in the conserved structure, particularly near the distal helix F.     

Mechanism of titin unfolding by force: insight from quasi-equilibrium molecular dynamics calculations

We have studied the unfolding by force of one of the immunoglobulin domains of the muscle protein titin using molecular dynamics simulations at 300 K. Previous studies, done at constant pulling rates, showed that under the effect of the force two strands connected to each other by six backbone H-bonds are pulled apart. No details about the mechanism of H-bond breaking were provided. Our simulation protocol "pull and wait" was designed to correspond to very slow pulling, more similar to the rates used in experiments than are the protocols used in previous computational studies. Under these conditions interstrand backbone H-bonds are not "ripped apart" by the application of the force. Instead, small elongations produced by the force weaken specific backbone H-bonds with respect to water-backbone H-bonds. These weakened bonds allow a single water molecule to make H-bonds to the CO and the NH of the same backbone H-bond while they are still bound to each other. The backbone H-bond then breaks (distance > 3.6 A), but its donor and acceptor atoms remain bound to the same water molecule. Further separation of the chains takes place when a second water molecule makes an H-bond with either the protein backbone donor or acceptor atom. Thus, the force does not directly break the main chain H-bonds: it destabilizes them in such a way that they are replaced by H-bonds to water. With this mechanism, the force necessary to break all the H-bonds required to separate the two strands will be strongly dependent on the pulling speed. Further simulations carried out at low forces but long waiting times (> or = 500 ps, < or = 10 ns) show that, given enough time, even a very small pulling force (< 400 pN) is sufficient to destabilize the interstrand H-bonds and allow them to be replaced by H-bonds to two water molecules. As expected, increasing the temperature to 350 K allows the interstrand H-bonds to break at lower forces than those required at 300 K.     

On the biochemical classification of yeast trehalases: Candida albicans contains two enzymes with mixed features of neutral and acid trehalase activities

Two enzymes endowed with trehalase activity are present in Candida albicans. The cytosolic trehalase (Ntc1p), displayed high activity in exponential phase regardless of the carbon source (glucose, trehalose or glycerol). Ntc1p activity was similar in neutral (pH 7.1) or acid (pH 4.5) conditions, strongly inhibited by ATP, weakly stimulated by divalent cations (Ca²⁺or Mn²⁺) and unaffected in the presence of cyclic AMP. The Ntc1p activity decreased in stationary phase, except in glycerol-grown cultures, but the catalytic properties did not change. In turn, the cell wall-linked trehalase (Atc1p) showed elevated activity in resting cells or in cultures growing on trehalose or glycerol. Although Atc1p is subjected to glucose repression, exhaustion of glucose in itself did not increased the activity. Significant Atc1p values could also be measured at neutral or acid pH, but Atc1p was insensitive to ATP, cyclic AMP and divalent cations. These results are in direct contrast with the current classification of yeast trehalases based on their optimum pH. They are also relevant in the light of the proposed use of trehalase inhibitors for the treatment of candidiasis.     

Statistical analysis of atomic contacts at RNA-protein interfaces

Forty-five crystals of complexes between proteins and RNA molecules from the Protein Data Bank have been statistically surveyed for the number of contacts between RNA components (phosphate, ribose and the four bases) and amino acid side chains. Three groups of complexes were defined: the tRNA synthetases; the ribosomal complexes; and a third group containing a variety of complexes. The types of atomic contacts were a priori classified into ionic, neutral H-bond, C-H...O H-bond, or van der Waals interaction. All the contacts were organized into a relational database which allows for statistical analysis. The main conclusions are the following: (i) in all three groups of complexes, the most preferred amino acids (Arg, Asn, Ser, Lys) and the less preferred ones (Ala, Ile, Leu, Val) are the same; Trp and Cys are rarely observed (respectively 15 and 5 amino acids in the ensemble of interfaces); (ii) of the total number of amino acids located at the interfaces 22% are hydrophobic, 40% charged (positive 32%, negative 8%), 30% polar and 8% are Gly; (iii) in ribosomal complexes, phosphate is preferred over ribose, which is preferred over the bases, but there is no significant preference in the other two groups; (iv) there is no significant prevalence of a base type at protein-RNA interfaces, but specifically Arg and Lys display a preference for phosphate over ribose and bases; Pro and Asn prefer bases over ribose and phosphate; Met, Phe and Tyr prefer ribose over phosphate and bases. Further, Ile, Pro, Ser prefer A over the others; Leu prefers C; Asp and Gly prefer G; and Asn prefers U. Considering the contact types, the following conclusions could be drawn: (i) 23% of the contacts are via potential H-bonds (including CH...O H-bonds and ionic interactions), 72% belong to van der Waals interactions and 5% are considered as short contacts; (ii) of all potential H-bonds, 54% are standard, 33% are of the C-H...O type and 13% are ionic; (iii) the Watson-Crick sites of G, O6(G) and principally N2(G) and the hydroxyl group O2' is more often involved in H-bonds than expected; the protein main chain is involved in 32% and the side chains in 68% of the H-bonds; considering the neutral and ionic H-bonds, the following couples are more frequent than expected-base A-Ser, base G-Asp/Glu, base U-Asn. The RNA CH groups interact preferentially with oxygen atoms (62% on the main chain and 19% on the side chains); (iv) the bases are involved in 38% of all H-bonds and more than 26% of the H-bonds have the H donor group on the RNA; (v) the atom O2' is involved in 21% of all H-bonds, a number greater than expected; (vi) amino acids less frequently in direct contact with RNA components interact frequently via their main chain atoms through water molecules with RNA atoms; in contrast, those frequently observed in direct contact, except Ser, use instead their side chain atoms for water bridging interactions.     

克鲁氏假丝酵母在高渗环境中海藻糖和胞内甘油积累的研究

Brown[1]在1976年提出了相容溶质(Compatible solutes)的概念,尽管有关它们功能的确切机制尚不是非常清楚,但是通常它们被认为是具有渗透调节作用和对细胞中生物活性物质具保护功能的物质.海藻糖和甘油在这方面所表现出的特殊功能已被国内外广泛关注[2].Brown[3]和Crowe[4]还分别报道了甘油和海藻糖在保护胞内可溶性酶和细胞膜稳定性方面的功能.Crowe[5]在研究几种不同碳水化合物对动物肌细胞的保护功能时发现,海藻糖和甘油都在不同程度上表现出这种特性.关于酵母细胞在加盐培养基中的生长代谢情况Kuniho Nakata[6]和Sukesh [7]分别进行了报道,发现酵母细胞内有海藻糖的积累,并且海藻糖的量与细胞对外界不利环境的耐受性有密切关系.     

Long-term molecular dynamics simulation of copper plastocyanin in water

A long molecular dynamics simulation (1.1 ns) of fully hydrated plastocyanin has been performed and analysed to relate protein dynamics to structural elements and functional properties. The solvated structure is described in detail by the analysis of H-bond network. During all the simulation, the crystal H-bond network is maintained in the beta-sheet regions, while several H-bonds are broken or formed on the external surface of the protein. To evaluate whether such changes could be due to conformational rearrangements or to solvent competition, we have examined the average number of H-bonds between protein atoms and water molecules, and the root mean square deviations from crystal structure as a function of protein residues. Protein mobility and flexibility have been examined by positional and dihedral angle rms fluctuations. Finally, cross-correlation maps have revealed the existence of correlated motions among residues connected by hydrogen bonds.     

Heterologous expression of mammalian Na/H antiporters in Saccharomyces cerevisiae

Na⁺/H⁺ antiporters, integral membrane proteins that exchange protons for alkali metal cations, play multiple roles in probably all living organisms (preventing cells from excessive amounts of alkali metal cations, regulating intracellular pH and cell volume). In this work, we studied the functionality of rat plasma membrane NHE1–3 exchangers upon their heterologous expression in alkali-metal-cation sensitive Saccharomyces cerevisiae, and searched for conditions that would increase their level in the plasma membrane and improve their functionality. Though three tested exchangers were partially localized to the plasma membrane (and two of them (NHE2 and NHE3) in an active form), the bulk of the synthesized proteins were arrested along the secretory pathway, mainly in the ER. To increase the level of exchangers in the yeast plasma membrane several approaches (truncation of C-terminal regulatory sequences, expression in mutant yeast strains, construction of rat/yeast protein chimeras, various growth conditions and chemical chaperones) were tested. The only increase in the amount of NHE exchangers in the plasma membrane was obtained upon expression in a strain with the npi1 mutation, which significantly lowers the level of Rsp5 ubiquitin ligase in cells. This mutation helped to stabilize proteins in the plasma membrane.     

Influence of steroids on hydrogen bonds in membranes assessed by near infrared spectroscopy

The covalent OH bonds of water vibrate and absorb radiation in the near infrared (NIR) region at wavelengths that vary according to the strength of the bonds which, at the same time, are sensitive to the number and/or strength of hydrogen bonds. By means of multivariate analytical tools, such spectral shift was exploited to study the effect of temperature, 25-hydroxycholesterol and progesterone on the H-bonded network of water in DMPA membranes. Temperature was found as the dominating factor altering the NIR spectra of water and then the H-bonds. Increasing temperatures disrupt the H-bonds network, strengthening the OH covalent bonds. The disruption of the H-bonds along the 13–58 °C range was noticeably greater than that caused by lipids or steroids at 500 μM. The H-bonded network of the interfacial water in DMPA membranes was disrupted by the presence of 25-hydroxycholesterol, but no significant disruption was observed in the presence of progesterone. The reduction of the H-bonds entails a reduction in the aggregation of the interfacial water by a reduction in the number of H-bonded molecules. It is proposed that the number of water molecules bonded with two H-bonds diminishes and the number of molecules with no H-bond increases roughly at similar proportions, with a constant population of molecules with one H-bond. The opposed effects of steroids are discussed in the context of their opposed effects on the phase state of membranes, the membrane water content and the steroid molecular structure.     

The key event in force-induced unfolding of Titin's immunoglobulin domains

Steered molecular dynamics simulation of force-induced titin immunoglobulin domain I27 unfolding led to the discovery of a significant potential energy barrier at an extension of approximately 14 A on the unfolding pathway that protects the domain against stretching. Previous simulations showed that this barrier is due to the concurrent breaking of six interstrand hydrogen bonds (H-bonds) between beta-strands A' and G that is preceded by the breaking of two to three hydrogen bonds between strands A and B, the latter leading to an unfolding intermediate. The simulation results are supported by Angstrom-resolution atomic force microscopy data. Here we perform a structural and energetic analysis of the H-bonds breaking. It is confirmed that H-bonds between strands A and B break rapidly. However, the breaking of the H-bond between strands A' and G needs to be assisted by fluctuations of water molecules. In nanosecond simulations, water molecules are found to repeatedly interact with the protein backbone atoms, weakening individual interstrand H-bonds until all six A'-G H-bonds break simultaneously under the influence of external stretching forces. Only when those bonds are broken can the generic unfolding take place, which involves hydrophobic interactions of the protein core and exerts weaker resistance against stretching than the key event.     

Hydrogen bonding interactions in noradrenaline-DMSO complexes: DFT and QTAIM studies of structure,properties and topology

The hydrogen bonding interactions between noradrenaline (NA) and DMSO were studied with density functional theory (DFT) regarding their geometries, energies, vibrational frequencies, and topological features of the electron density. The quantum theory of atoms in molecules (QTAIM) and the natural bond orbital (NBO) analyses were employed to elucidate the hydrogen bonding interaction characteristics in noradrenaline-DMSO complexes. The H-bonds involving the hydroxyls hydrogen in NA and the O atom in DMSO are dominant intermolecular H-bonds and are stronger than other H-bonds involving the methyl hydrogen of DMSO as a H-donor. The weak H-bonds also include a π H-bond which involves the benzene ring as a H-donor or H-acceptor. QTAIM identified the weak H-bonds formed between the methyl hydrogen of DMSO and the N atom in NA in some complexes (AB5, AB6 and AB7), which cannot be further confirmed by NBO and other methods, so there are probably no interactions between hydrogen and nitrogen atoms among these complexes. A good linear relationship between logarithmic electron density (lnρ _b ) at the bond critical point (BCP) and structural parameter (δR _H···Y) was found. The formations of new H-bonds in some complexes are helpful to strengthen the original intramolecular H-bond, this is attributed to the cooperativity of H-bonds in complexes and can be learned from the structure results and the NBO and QTAIM analyses. Analysis of various physically meaningful contributions arising from the energy decomposition procedures show that the orbital interactions of H-bond is predominant during the formation of the complex, moreover, both the hydrogen bonding interaction and the structural deformation are responsible for the stability of the complexes.     

Examining water in model membranes by near infrared spectroscopy and multivariate analysis

By exploiting the sensitivity of the NIR spectrum, particularly the first overtone of water, to the number and strength of hydrogen bonds, the hydrogen bond network and water polymerization in membranes of DMPA (1,2-dimyristoyl-sn-glycero-3-phosphate) and DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) was investigated as a function of the temperature and the presence of this two phospholipids having the same tail but different polar head. Principal components analysis performed on the spectra was used to disclose subtle spectral changes that mirror the alteration of the vibrational energy of the water O-H bonds, as a measure of the H-bond network. Temperature showed a dominating effect on the H-bond network. Increasing temperatures diminished the number of strongly H-bonded water molecules and increased the number of weakly H-bonded waters. This main effect of temperature was missing after the subtraction of the pure water spectra from the lipid-containing ones. An intriguing secondary effect of temperature was also revealed. Phospholipids exhibited an effect qualitatively similar to that of the temperature. DMPA, and particularly DMPC, disrupted the H-bond network in the neighboring lipid-water interface, reducing water polymerization and strengthening the water O-H bonds. The type of the polar head affects the H-bonds more than duplicate the concentration of the lipid. A connection between head group structure and the effect on the H-bonds network, and the existence of two populations of water molecules are discussed.     

Infrared absorption study of the heme pocket dynamics of carbonmonoxyheme proteins

The temperature dependencies of the infrared absorption CO bands of carboxy complexes of horseradish peroxidase (HRP(CO)) in glycerol/water mixture at pH 6.0 and 9.3 are interpreted using the theory of optical absorption bandshape. The bands' anharmonic behavior is explained assuming that there is a higher-energy set of conformational substates (CSS(h)), which are populated upon heating and correspond to the protein substates with disordered water molecules in the heme pocket. Analysis of the second moments of the CO bands of the carboxy complexes of myoglobin (Mb(CO)) and hemoglobin (Hb(CO)), and of HRP(CO) with benzohydroxamic acid (HRP(CO)+BHA), shows that the low energy CSS(h) exists also in the open conformation of Mb(CO), where the heme pocket is spacious enough to accommodate a water molecule. In the HRP(CO)+BHA and closed conformations of Mb(CO) and Hb(CO), the heme pocket is packed with BHA and different amino acids, the CSS(h) has much higher energy and is hardly populated even at the highest temperatures. Therefore only motions of these amino acids contribute to the band broadening. These motions are linked to the protein surface and frozen in the glassy matrix, whereas in the liquid solvent they are harmonic. Thus the second moment of the CO band is temperature-independent in glass and is proportional to the temperature in liquid. The temperature dependence of the second moment of the CO peak of HRP(CO) in the trehalose glass exhibits linear coupling to an oscillator. This oscillator can be a moving water molecule locked in the heme pocket in the whole interval of temperatures or a trehalose molecule located in the heme pocket.     

Glycerol inhibits water permeation through Plasmodium Falciparum aquaglyceroporin

Plasmodium falciparum aquaglyceroporin (PfAQP) is a multifunctional membrane protein in the plasma membrane of P. falciparum, the parasite that causes the most severe form of malaria. The current literature has established the science of PfAQP’s structure, functions, and hydrogen-bonding interactions but left unanswered the following fundamental question: does glycerol modulate water permeation through aquaglyceroporin that conducts both glycerol and water? This paper provides an affirmative answer to this question of essential importance to the protein’s functions. On the basis of the chemical-potential profile of glycerol from the extracellular bulk region, throughout PfAQP’s conducting channel, to the cytoplasmic bulk region, this study shows the existence of a bound state of glycerol inside aquaglyceroporin’s permeation pore, from which the dissociation constant is approximately 14 μM. A glycerol molecule occupying the bound state occludes the conducting pore through which permeating molecules line up in single file by hydrogen-bonding with one another and with the luminal residues of aquaglyceroporin. In this way, glycerol inhibits permeation of water and other permeants through aquaglyceroporin. The biological implications of this theory are discussed and shown to agree with the existent in vitro data. It turns out that the structure of aquaglyceroporin is perfect for the van der Waals interactions between the protein and glycerol to cause the existence of the bound state deep inside the conducting pore and, thus to play an unexpected but significant role in aquaglyceroporin’s functions.