Successful design and synthesis of a polarity-triggered beta-->alpha conformational switch using the side chain interaction index (SCII) as a measure of local structural stability

Certain sequences within proteins have the ability to undergo an abrupt cooperative conformational switch from beta-strand to helix in response to decreasing polarity of the environment. This behavior was first observed at the CD4 binding site of the envelope glycoprotein gp120 of HIV-1, but evidence has accumulated that polarity-driven beta --> alpha switches may be widespread, serving both to facilitate binding on protein/membrane or protein/protein contact and to signal that docking has occurred. The characteristics identified so far that distinguish switch sequences (a reverse turn at the N-terminus that acts as a helix initiation site, a conserved tryptophan residue downstream, and high potential for both the helix and beta-fold) appear to be necessary but not sufficient, as some otherwise promising sequences found in data bank searches proved not to be capable of cooperative refolding. Analysis of existing switches has led to the development of the side chain interaction index (SCII) as a further parameter characterizing the beta --> alpha polarity-driven switch. Data bank searches using this additional parameter have successfully identified a series of new potential switch sequences. All of them have in common the amino acid tetrad LPCR at the N-terminus and a tryptophan 5-20 residues C-terminal to it. Those with a high SCII as well, when synthesized and tested, exhibited strongly cooperative polarity-driven refolding. Control peptides, containing all other parameters but with a low SCII, did not. Using this new information, an artificial sequence was designed that had a high SCII as well as the initiation site, conserved tryptophan, and high Palpha and Pbeta. When synthesized and tested, this sequence did in fact behave as a conformational switch, refolding cooperatively from beta-fold to helix at a threshold value of 30% TFE. The successful design of a polarity-driven conformational switch opens the possibility of using this motif as a tool in protein engineering.     

Is Asp‐His‐Ser/Thr‐Trp tetrad hydrogen‐bond network important to WD40‐repeat proteins: A statistical and theoretical study

WD40‐repeat proteins are abundant and play important roles in forming protein complexes. The domain usually has seven WD40 repeats, which folds into a seven β‐sheet propeller with each β‐sheet in a four‐strand structure. An analysis of 20 available WD40‐repeat proteins in Protein Data Bank reveals that each protein has at least one Asp‐His‐Ser/Thr‐Trp (D‐H‐S/T‐W) hydrogen‐bonded tetrad, and some proteins have up to six or seven such tetrads. The relative positions of the four residues in the tetrads are also found to be conserved. A sequence alignment analysis of 560 WD40‐repeat protein sequences in human reveals very similar features, indicating that such tetrad may be a general feature of WD40‐repeat proteins. We carried out density functional theory and found that these tetrads can lead to significant stabilization including hydrogen‐bonding cooperativity. The hydrogen bond involving Trp is significant. These results lead us to propose that the tetrads may be critical to the stability and the mechanism of folding of these proteins. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Solvent polarity-dependent structural refolding: A CD and NMR study of a 15 residue peptide

A close association between the HIV surface protein gp120 and the CD4 T cell receptor initiates the viral multiplication cycle. A 15 amino acid peptide (LAV) within the CD4 binding domain of gp 120 has been shown to retain receptor binding ability. The structural behavior of the LAV peptide has been studied by CD and NMR methods in aqueous solution and upon addition of trifluoroethanol (TFE) to emulate the relatively apolar conditions at the membrane bound receptor. Previous work has shown that the LAV peptide folds into a β-pleated structure in more polar buffer/TFE mixtures, while a concerted structural change can be observed at a concentration of 60% TFE (v/v). This abrupt, cooperative refolding from a regular β-sheet to a helical secondary structure is known as “switch” behavior. Former CD experiments with LAV sequence variants have supported the assumption that four amino acids at the N-terminus (LPCR) are indispensable for the “switch.” The tetrad has a strong β-turn forming potential. The suggestion has been formulated that the tetrad can act as a nucleation site governing the refolding. The present NMR study of the LAV peptide in TFE gives evidence for a 3₁₀-helix suggesting that the tetrad adopts a type III β-turn and promotes the formation of a similar bend in the next overlapping tetrad until the sequence is restructured into a 3₁₀-helix at a critical polarity favoring intrachain hydrogen bonds. © 1995 Wiley-Liss, Inc.     

Molecular dynamics study of the proposed beta-hairpin form of the switch domain from HIV1 gp120 alone and complexed with an inhibitor of CD4 binding

The strong tendency of beta-hairpin peptides to aggregate can prevent their structural resolution. The polar form of the switch peptide (LAV 15mer) at the CD4-binding domain of HIV1 gp120 is such a peptide, and NMR investigations of its interaction with a class of CD4-binding inhibitors developed in this laboratory have been hindered. Detailed knowledge of the interaction is required for the development of more potent switch inhibitors, that act by disrupting the cooperative folding transition necessary for binding to the CD4 receptor. In carrying out molecular dynamics simulation of the free peptide under polar conditions, we found that the properties of the resulting structure agree closely with those observed by circular dichroism. The same conditions, used to model the peptide/ inhibitor complex, produced a stable bimolecular structure with specific interactions between the inhibitor and side chains on the peptide, (e.g., Trp12 and the LPCR tetrad), known to control the folding transition. These help explain existing data on the relative potency of inhibitor derivatives and provide a basis for improved inhibitor design.     

Functional evolution of the HIV-1 envelope glycoprotein 120 association site of glycoprotein 41

Protein-protein interaction surfaces can exhibit structural plasticity, a mechanism whereby an interface adapts to mutations as binding partners coevolve. The HIV-1 envelope glycoprotein gp120-gp41 complex, which is responsible for receptor attachment and membrane fusion, represents an extreme example of a coevolving complex as up to 35% amino acid sequence divergence has been observed in these proteins among HIV-1 isolates. In this study, the function of conserved gp120 contact residues, Leu593, Trp596, Gly597, Lys601, and Trp610 within the disulfide-bonded region of gp41, was examined in envelope glycoproteins derived from diverse HIV-1 isolates. We found that the gp120-gp41 association function of the disulfide-bonded region is conserved. However, the contribution of individual residues to gp41 folding and/or stability, gp120-gp41 association, membrane fusion function, and viral entry varied from isolate to isolate. In gp120-gp41 derived from the dual-tropic isolate, HIV-189.6, the importance of Trp596 for fusion function was dependent on the chemokine receptor utilized as a fusion cofactor. Thus, the engagement of alternative chemokine receptors may evoke distinct fusion-activation signals involving the site of gp120-gp41 association. An examination of chimeric glycoproteins revealed that the isolate-specific functional contributions of particular gp120-contact residues are influenced by the sequence of gp120 hypervariable regions 1, 2, and 3. These data indicate that the gp120-gp41 association site is structurally and functionally adaptable, perhaps to maintain a functional glycoprotein complex in a setting of host selective pressures driving the rapid coevolution of gp120 and gp41.     

ULTRASTRUCTURE OF SPOROGENESIS IN THE MOSS,AMBLYSTEGIUM RIPARIUM I. MEIOSIS AND CYTOKINESIS

Emphasis is placed on three aspects of meiosis in the moss Amblystegium riparium (Hedw.) BSG: 1***) nature of the sporogenous layer; 2) prophasic microtubules and polarity; and 3) cleavage pattern. Spore tetrads develop while still encased by archesporial cell walls. The cellular nature of the sporogenous layer differs from the more usual occurrence of free sporocytes released into a common spore sac. Two important events mark the establishment of sporocyte polarity during meiotic prophase: 1) migration of the four plastids to the distal tetrad poles (telophase II poles); and 2) ingrowth of the sporocyte wall in eventual cleavage planes between the tetrad poles. An extensive, plastid-based microtubule system is associated with organelle migration during the establishment of sporocyte polarity in meiotic prophase. Disruption of the nuclear envelope in prometaphase I occurs at sites opposite the four plastids where microtubules extend from plastid envelope to nuclear envelope. Formation of a cell plate following the first meiotic division results in a dyad, whereas in many mosses meiosis is completed in the undivided sporocyte and is followed by simultaneous cleavage into a spore tetrad. Spore cleavage is accomplished by vesicular coalescence resulting in septa that coincide with the prophasic wall ingrowths.     

Specific interactions of sticholysin I with model membranes: An NMR study

Sticholysin I (StnI) is an actinoporin produced by the sea anemone Stichodactyla helianthus that binds biological and model membranes forming oligomeric pores. Both a surface cluster of aromatic rings and the N‐terminal region are involved in pore formation. To characterize the membrane binding by StnI, we have studied by ¹H‐NMR the environment of these regions in water and in the presence of membrane‐mimicking micelles. Unlike other peptides from homologous actinoporins, the synthetic peptide corresponding to residues 1–30 tends to form helix in water and is more helical in either trifluoroethanol or dodecylphosphocholine (DPC) micelles. In these environments, it forms a helix‐turn‐helix motif with the last α‐helical segment matching the native helix‐α₁ (residues 14–24) present in the complete protein. The first helix (residues 4–9) is less populated and is not present in the water‐soluble protein structure. The characterization of wild‐type StnI structure in micelles shows that the helix‐α₁ is maintained in its native structure and that this micellar environment does not provoke its detachment from the protein core. Finally, the study of the aromatic resonances has shown that the motional flexibility of specific rings is perturbed in the presence of micelles. On these bases, the implication of the aromatic rings of Trp‐111, Tyr‐112, Trp‐115, Tyr‐132, Tyr‐136, and Tyr‐137, in the interaction between StnI and the micelle is discussed. Based on all the findings, a revised model for StnI interaction with membranes is proposed, which accounts for differences in its behavior as compared with other highly homologous sticholysins. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Lactose repressor hinge domain independently binds DNA

The short 8–10 amino acid “hinge” sequence in lactose repressor (LacI), present in other LacI/GalR family members, links DNA and inducer‐binding domains. Structural studies of full‐length or truncated LacI‐operator DNA complexes demonstrate insertion of the dimeric helical “hinge” structure at the center of the operator sequence. This association bends the DNA ～40° and aligns flanking semi‐symmetric DNA sites for optimal contact by the N‐terminal helix‐turn‐helix (HtH) sequences within each dimer. In contrast, the hinge region remains unfolded when bound to nonspecific DNA sequences. To determine ability of the hinge helix alone to mediate DNA binding, we examined (i) binding of LacI variants with deletion of residues 1–50 to remove the HtH DNA binding domain or residues 1–58 to remove both HtH and hinge domains and (ii) binding of a synthetic peptide corresponding to the hinge sequence with a Val52Cys substitution that allows reversible dimer formation via a disulfide linkage. Binding affinity for DNA is orders of magnitude lower in the absence of the helix‐turn‐helix domain with its highly positive charge. LacI missing residues 1–50 binds to DNA with ～4‐fold greater affinity for operator than for nonspecific sequences with minimal impact of inducer presence; in contrast, LacI missing residues 1–58 exhibits no detectable affinity for DNA. In oxidized form, the dimeric hinge peptide alone binds to O1 and nonspecific DNA with similarly small difference in affinity; reduction to monomer diminished binding to both O1 and nonspecific targets. These results comport with recent reports regarding LacI hinge interaction with DNA sequences.     

Structural requirements for gp80 independence of human herpesvirus 8 interleukin-6 (vIL-6) and evidence for gp80 stabilization of gp130 signaling complexes induced by vIL-6

Human herpesvirus 8 interleukin-6 (vIL-6) displays 25% amino acid identity with human IL-6 (hIL-6) and shares an overall four-helix-bundle structure and gp130-mediated STAT/mitogen-activated protein kinase signaling with its cellular counterpart. However, vIL-6 is distinct in that it can signal through gp130 alone, in the absence of the nonsignaling gp80 alpha-subunit of the IL-6 receptor. To investigate the structural requirements for gp80 independence of vIL-6, a series of expression vectors encoding vIL-6/hIL-6 chimeric and site-mutated IL-6 proteins was generated. The replacement of hIL-6 residues with three vIL-6-specific tryptophans implicated in gp80 independence from crystallographic studies or the A and C helices containing these residues did not confer gp80 independence to hIL-6. The N- and C-terminal regions of vIL-6 could be substituted with hIL-6 sequences with the retention of gp80-independent signaling, but substitutions of other regions of vIL-6 (helix A, A/B loop, helix B, helix C, and proximal half of helix D) with equivalent sequences of hIL-6 abolished gp80 independence. Interestingly, the B helix of vIL-6 was absolutely required for gp80 independence, despite the fact that this region contains no receptor-binding residues. Point mutational analysis of helix C, which contains residues involved in physical and functional interactions with gp130 domains 2 and 3 (cytokine-binding homology region), identified a variant, VI120EE, that was able to signal and dimerize gp130 only in the presence of gp80. gp80 was also found to stabilize gp130:g130 dimers induced by a distal D helix variant of vIL-6 that was nonetheless able to signal independently of gp80. Together, our data reveal the crucial importance of overall vIL-6 structure and conformation for gp80-independent signaling and provide functional and physical evidence of the stabilization of vIL-6-induced gp130 signaling complexes by gp80.     

Structural and mutational studies on an aldo‐keto reductase AKR5C3 from Gluconobacter oxydans

An aldo‐keto reductase AKR5C3 from Gluconobacter oxydans (designated as Gox0644) is a useful enzyme with various substrates, including aldehydes, diacetyl, keto esters, and α‐ketocarbonyl compounds. The crystal structures of AKR5C3 in apoform in complex with NADPH and the D53A mutant (AKR5C3^‐D53A) in complex with NADPH are presented herein. Structure comparison and site‐directed mutagenesis combined with biochemical kinetics analysis reveal that the conserved Asp53 in the AKR5C3 catalytic tetrad has a crucial role in securing active pocket conformation. The gain‐of‐function Asp53 to Ala mutation triggers conformational changes on the Trp30 and Trp191 side chains, improving NADPH affinity to AKR5C3, which helps increase catalytic efficiency. The highly conserved Trp30 and Trp191 residues interact with the nicotinamide moiety of NADPH and help form the NADPH‐binding pocket. The AKR5C3^‐W30A and AKR5C3^‐W191Y mutants show decreased activities, confirming that both residues facilitate catalysis. Residue Trp191 is in the loop structure, and the AKR5C3^‐W191Y mutant does not react with benzaldehyde, which might also determine substrate recognition. Arg192, which is involved in the substrate binding, is another important residue. The introduction of R192G increases substrate‐binding affinity by improving hydrophobicity in the substrate‐binding pocket. These results not only supplement the AKRs superfamily with crystal structures but also provide useful information for understanding the catalytic properties of AKR5C3 and guiding further engineering of this enzyme.     

Correlated mutations at gp120 positions 322 and 440: implications for gp120 structure

Analysis of V3 and C4 sequences of HIV-1 reveals correlated mutations at gp120 positions 322 and 440, and a very strong preference for a positively charged residue at position 440 when position 322 is negatively charged. This observation suggests that these two residues are close to each other and interact electrostatically in R5 viruses. This interaction was used to model V3 in the context of gp120 using NMR data for the V3 loop and the crystal structure of the gp120-core. The interaction between residues 322 and 440 may serve as part of the molecular switch for HIV-1 phenotype conversion.     

Dimeric DNA quadruplex containing major groove-aligned A-T-A-T and G-C-G-C tetrads stabilized by inter-subunit Watson-Crick A-T and G-C pairs

We report on an NMR study of unlabeled and uniformly 13C,15N-labeled d(GAGCAGGT) sequence in 1 M NaCl solution, conditions under which it forms a head-to-head dimeric quadruplex containing sequentially stacked G-C-G-C, G-G-G-G and A-T-A-T tetrads. We have identified, for the first time, a slipped A-T-A-T tetrad alignment, involving recognition of Watson-Crick A-T pairs along the major groove edges of opposing adenine residues. Strikingly, both Watson-Crick G-C and A-T pairings within the direct G-C-G-C and slipped A-T-A-T tetrads, respectively, occur between rather than within hairpin subunits of the dimeric d(GAGCAGGT) quadruplex. The hairpin turns in the head-to-head dimeric quadruplex involve single adenine residues and adds to our knowledge of chain reversal involving edgewise loops in DNA quadruplexes. Our structural studies, together with those from other laboratories, definitively establish that DNA quadruplex formation is not restricted to G(n) repeat sequences, with their characteristic stacked uniform G-G-G-G tetrad architectures. Rather, the quadruplex fold is a more versatile and robust architecture, accessible to a range of mixed sequences, with the potential to facilitate G-C-G-C and A-T-A-T tetrad through major and minor groove alignment, in addition to G-G-G-G tetrad formation. The definitive experimental identification of such major groove-aligned mixed A-T-A-T and G-C-G-C tetrads within a quadruplex scaffold, has important implications for the potential alignment of duplex segments during homologous recombination.     

Mapping of the interaction between the immunodominant loop of the ectodomain of HIV-1 gp41 and human complement protein C1q.

The human immunodeficiency virus type 1 transmembrane envelope glycoprotein gp41 has been previously shown to activate the C1 complex of human complement through direct interaction with its C1q subunit. The major interaction site has been located within the gp41 immunodominant region (residues 590-620), and a synthetic peptide overlapping residues 601-613 of gp41 (sequence GIWGCSGKLICTT) was shown to inhibit binding of gp41 to C1q in vitro (Thielens, N.M., Bally, I.M., Ebenbichler, C.F., Dierich, M.P. & Arlaud, G.J. (1993) J. Immunol. 151, 6583-6592). The ectodomain of gp41 (s-gp41) was secreted from the methylotrophic yeast Pichia pastoris and purified by immunoaffinity chromatography. Enzymatic deglycosylation of the recombinant s-gp41 was necessary to allow its in vitro interaction with C1q. A solid-phase competition assay was used to monitor the effect of mutant peptides derived from segment 601-613 of gp41 on the binding of deglycosylated s-gp41 to C1q. Whereas mutation of Ser606 had no effect, replacement of Ile602, Trp603, Lys608, Leu609 and Ile610 by Ala abolished the ability of the resulting peptides to inhibit binding of s-gp41 to C1q, suggesting that these residues participate in the interaction between gp41 and C1q. These findings are discussed in the light of a structural model of the immunodominant loop of gp41. It is proposed that the recognition of gp41 by C1q is driven by hydrophobic interactions, and that the sites of gp41 responsible for interaction with gp120 and C1q partly overlap.     

Epitope Mapping of M36, a Human Antibody Domain with Potent and Broad HIV-1 Inhibitory Activity

M36 is the first member of a novel class of potent HIV-1 entry inhibitors based on human engineered antibody domains (eAds). It exhibits broad inhibitory activity suggesting that its CD4-induced epitope is highly conserved. Here, we describe fine mapping of its epitope by using several approaches. First, a panel of mimotopes was affinity-selected from a random peptide library and potential m36-binding residues were computationally predicted. Second, homology modeling of m36 and molecular docking of m36 onto gp120 revealed potentially important residues in gp120-m36 interactions. Third, the predicted contact residues were verified by site-directed mutagenesis. Taken together, m36 epitope comprising three discontinuous sites including six key gp120 residues (Site C1: Thr123 and Pro124; Site C3: Glu370 and Ile371; Site C4: Met426 and Trp427) were identified. In the 3D structure of gp120, the sites C1 and C4 are located in the bridging sheet and the site C3 is within the β15-α3 excursion, which play essential roles for the receptor- and coreceptor-binding and are major targets of neutralizing antibodies. Based on these results we propose a precise localization of the m36 epitope and suggest a mechanism of its broad inhibitory activity which could help in the development of novel HIV-1 therapeutics based on eAds.     

Reduction in vacuolar volume in the tapetal cells coincides with conclusion of the tetrad stage in Arabidopsis thaliana

Although the tapetum is known for its role in the removal of the tetrad wall, a morphological change in the tapetum correlating with such a role has not been described. Here we report that in two ecotypes of Arabidopsis thaliana, Landsberg erecta and Columbia, the vacuoles in tapetal cells underwent progressive enlargement prior to the separation of tetrads but became drastically reduced when tetrads just separated from one another. Such a drastic change in vacuolar volume was not observed in later anther development. We also observed that the walls of associated tetrads were much less stained with Toluidine Blue O than the walls of separate tetrads, indicating that the tetrad walls underwent an alteration during the tetrad stage. Furthermore, we identified the N-terminal propeptide signals for sorting vacuolar proteins in 15 β-1,3-glucanases, five polygalacturonases, and two endocellulases that are expressed in Arabidopsis young floral buds; all three types of the enzymes are known to participate in degradation of the tetrad wall. These results suggest that the tapetal vacuoles might be a storage site for these enzymes prior to their secretion to the anther locule.     

HIV-1 envelope accessible surface and polarity: clade, blood, and brain

The human immunodeficiency virus type-1 (HIV-1) gp160 (gp120-gp41 complex) trimer envelope (ENV) protein is a potential vaccine candidate for HIV/AIDS. HIV-1 vaccine development has been problematic and charge polarity as well as sequence variation across clades may relate to the difficulties. Further obstacles are caused by sequence variation between blood and brain-derived sequences, since the brain is a separate compartment for HIV-1 infection. We utilize a threedimensional residue measure of solvent exposure, accessible surface area (ASA), which shows that major segments of gp120 and gp41 known structures are solvent exposed across clades. We demonstrate a large percent sequence polarity for solvent exposed residues in gp120 and gp41. The range of sequence polarity varies across clades, blood, and brain from different geographical locations. Regression analysis shows that blood and brain gp120 and gp41 percent sequence polarity range correlate with mean Shannon entropy. These results point to the use of protein modifications to enhance HIV-1 ENV vaccines across multiple clades, blood, and brain. It should be noted that we do not address the issue of protein glycosylation here; however, this is an important issue for vaccine design and development. ABBREVIATIONS: HIV-1 - human immunodeficiency virus type 1, AIDS - acquired immunodeficiency syndrome, ENV - envelope, gp160 - 160,000d glycoprotein, gp120 - 120,000d glycoprotein, gp41 - 41,000d glycoprotein, LANL - Los Alamos National Laboratories, PDB - Protein Data Bank, HVTN - STEP HIV vaccine trial, AA - amino acids, MSA - multiple sequence alignment, ASA - accessible surface area, SNPs- single nucleotide polymorphisms, HAART - Highly Active Antiretroviral Therapy, CCR5 - C-C chemokine receptor type 5, CNS - central nervous system, HIVE - HIV encephalitis, P - polarity, NP - non-polarity, CTL - cytotoxic T lymphocyte, NIAID - National Institute of Allergy and Infectious Diseases.     

Structural and Functional Studies of the Phage Sf6 Terminase Small Subunit Reveal a DNA-Spooling Device Facilitated by Structural Plasticity

In many DNA viruses, genome packaging is initiated by the small subunit of the packaging terminase, which specifically binds to the packaging signal on viral DNA and directs assembly of the terminase holoenzyme. We have experimentally mapped the DNA-interacting region on Shigella virus Sf6 terminase small subunit gp1, which occupies extended surface areas encircling the gp1 octamer, indicating that DNA wraps around gp1 through extensive contacts. High‐resolution structures reveal large-scale motions of the gp1 DNA-binding domain mediated by the curved helix formed by residues 54–81 and an intermolecular salt bridge formed by residues Arg67 and Glu73, indicating remarkable structural plasticity underlying multivalent, pleomorphic gp1:DNA interactions. These results provide spatial restraints for protein:DNA interactions, which enable construction of a three-dimensional pseudo-atomic model for a DNA-packaging initiation complex assembled from the terminase small subunit and the packaging region on viral DNA. Our results suggest that gp1 functions as a DNA-spooling device, which may transform DNA into a specific architecture appropriate for interaction with and cleavage by the terminase large subunit prior to DNA translocation into viral procapsid. This may represent a common mechanism for the initiation step of DNA packaging in tailed double‐stranded DNA bacterial viruses.     

The effect of Asp-His-Ser/Thr-Trp tetrad on the thermostability of WD40-repeat proteins

We recently found that Asp-His-Ser/Thr-Trp hydrogen-bonded tetrads are widely and uniquely present in the WD40-repeat proteins. WDR5 protein is a seven WD40-repeat propeller with five such tetrads. To explore the effect of the tetrad on the structure and stability of WD40-repeat proteins, the wild-type WDR5 and its seven mutants involving the substitutions of tetrad residues have been isolated. The crystal structures of the wild-type WDR5 and its three WDR5 mutants have been determined by X-ray diffraction method. The mutations of the tetrad residues are found not to change the basic structural features. The denaturing profiles of the wild type and the seven mutants with the use of denaturant guanidine hydrochloride have been studied by circular dichroism spectroscopy to determine the folding free energies of these proteins. The folding free energies of the wild type and the S62A, S146A, S188A, D192E, W330F, W330Y, and D324E mutants are measured to be about -11.6, -2.7, -3.1, -2.9, -3.6, -7.1, -7.0, and -7.5 kcal/mol, respectively. These suggest that (1) the hydrogen bonds in these hydrogen bond networks are unusually strong; (2) each hydrogen-bonded tetrad provides over 12 kcal/mol stability to the protein; thus, the removal of any single tetrad would cause unfolding of the protein; (3) since there are five tetrads, the protein must be in a highly unstable state without the tetrads, which might be related to its biological functions.