The role of side chain conformational flexibility in surface recognition by Tenebrio molitor antifreeze protein

Two-dimensional nuclear magnetic resonance spectroscopy was used to investigate the flexibility of the threonine side chains in the beta-helical Tenebrio molitor antifreeze protein (TmAFP) at low temperatures. From measurement of the (3)J(alphabeta) (1)H-(1)H scalar coupling constants, the chi(1) angles and preferred rotamer populations can be calculated. It was determined that the threonines on the ice-binding face of the protein adopt a preferred rotameric conformation at near freezing temperatures, whereas the threonines not on the ice-binding face sample many rotameric states. This suggests that TmAFP maintains a preformed ice-binding conformation in solution, wherein the rigid array of threonines that form the AFP-ice interface matches the ice crystal lattice. A key factor in binding to the ice surface and inhibition of ice crystal growth appears to be the close surface-to-surface complementarity between the AFP and crystalline ice, and the lack of an entropic penalty associated with freezing out motions in a flexible ligand.     

Structure and dynamics of a beta-helical antifreeze protein

Antifreeze proteins (AFPs) protect many types of organisms from damage caused by freezing. They do this by binding to the ice surface, which causes inhibition of ice crystal growth. However, the molecular mechanism of ice binding leading to growth inhibition is not well understood. In this paper, we present the solution structure and backbone NMR relaxation data of the antifreeze protein from the yellow mealworm beetle Tenebrio molitor (TmAFP) to study the dynamics in the context of structure. The full (15)N relaxation analysis was completed at two magnetic field strengths, 500 and 600 MHz, as well as at two temperatures, 30 and 5 degrees C, to measure the dynamic changes that occur in the protein backbone at different temperatures. TmAFP is a small, highly disulfide-bonded, right-handed parallel beta-helix consisting of seven tandemly repeated 12-amino acid loops. The backbone relaxation data displays a periodic pattern, which reflects both the 12-amino acid structural repeat and the highly anisotropic nature of the protein. Analysis of the (15)N relaxation parameters shows that TmAFP is a well-defined, rigid structure, and the extracted parameters show that there is similar restricted internal mobility throughout the protein backbone at both temperatures studied. We conclude that the hydrophobic, rigid binding site may reduce the entropic penalty for the binding of the protein to ice. The beta-helical fold of the protein provides this rigidity, as it does not appear to be a consequence of cooling toward a physiologically relevant temperature.     

13C relaxation experiments for aromatic side chains employing longitudinal- and transverse-relaxation optimized NMR spectroscopy

Aromatic side chains are prevalent in protein binding sites, perform functional roles in enzymatic catalysis, and form an integral part of the hydrophobic core of proteins. Thus, it is of great interest to probe the conformational dynamics of aromatic side chains and its response to biologically relevant events. Indeed, measurements of (13)C relaxation rates in aromatic moieties have a long history in biomolecular NMR, primarily in the context of samples without isotope enrichment that avoid complications due to the strong coupling between neighboring (13)C spins present in uniformly enriched proteins. Recently established protocols for specific (13)C labeling of aromatic side chains enable measurement of (13)C relaxation that can be analyzed in a straightforward manner. Here we present longitudinal- and transverse-relaxation optimized pulse sequences for measuring R (1), R (2), and {(1)H}-(13)C NOE in specifically (13)C-labeled aromatic side chains. The optimized R (1) and R (2) experiments offer an increase in sensitivity of up to 35 % for medium-sized proteins, and increasingly greater gains are expected with increasing molecular weight and higher static magnetic field strengths. Our results highlight the importance of controlling the magnetizations of water and aliphatic protons during the relaxation period in order to obtain accurate relaxation rate measurements and achieve full sensitivity enhancement. We further demonstrate that potential complications due to residual two-bond (13)C-(13)C scalar couplings or dipolar interactions with neighboring (1)H spins do not significantly affect the experiments. The approach presented here should serve as a valuable complement to methods developed for other types of protein side chains.     

Improved labeling strategy for 13C relaxation measurements of methyl groups in proteins

Selective incorporation of ¹³C into the methyl groupsof protein side chains is described as a means for simplifying themeasurement and interpretation of ¹³C relaxation parameters.High incorporation (>90%) is accomplished by using pyruvate(3-¹³C, 99%) as the sole carbon source in the growthmedia for protein overexpression in E. coli. This improved labeling schemeincreases the sensitivity of the relaxation experiments by approximatelyfivefold when compared to randomly fractionally ¹³C-labeledprotein, allowing high-quality measurements on relatively dilute (<1 mM)protein samples at a relatively low cost.     

Carbon-13 NMR studies of the lysine side chains of calmodulin and its proteolytic fragments

ThepH-titration and dynamic behaviour of the seven lysine side chains in bovine calmodulin were studied by carbon-13 NMR. The amino groups of the calcium saturated protein and its proteolytic fragments TR₁C(1–75) and TR₂C (78–148) were dimethylated with carbon-13 labeled formaldehyde; this modification did not alter the protein's structure or its ability to activate the enzyme cyclic nucleotide phosphodiesterase. Tentative assignments for 5 out of the 7 dimethyl lysine resonances could be obtained by comparing spectra of the fully and partially modified protein, with those of the proteolytic fragments. ThepK_a values measured for calcium saturated calmodulin ranged between 9.5 (Lys 75) and 10.2 (Lys 13); two residues (Lys 94 and Lys 13) showed a biphasic titration curve suggesting their possible involvement in ion-pairs. The dynamic behavior of the lysine side chains was deduced from spin lattice relaxation measurements. All side chains were flexible and this was not influenced by the removal of calcium, or the addition of the calmodulin antagonist trifluoperazine. The latter data suggest that the lysine side chains are not directly involved in calmodulin's target binding sites.     

Tautomerism,acid-base equilibria,and H-bonding of the six histidines in subtilisin BPN' by NMR

We have determined by (15)N, (1)H, and (13)C NMR, the chemical behavior of the six histidines in subtilisin BPN' and their PMSF and peptide boronic acid complexes in aqueous solution as a function of pH in the range of from 5 to 11, and have assigned every (15)N, (1)H, C(epsilon 1), and C(delta2) resonance of all His side chains in resting enzyme. Four of the six histidine residues (17, 39, 67, and 226) are neutrally charged and do not titrate. One histidine (238), located on the protein surface, titrates with pK(a) = 7.30 +/- 0.03 at 25 degrees C, having rapid proton exchange, but restricted mobility. The active site histidine (64) in mutant N155A titrates with a pK(a) value of 7.9 +/- 0.3 and sluggish proton exchange behavior, as shown by two-site exchange computer lineshape simulation. His 64 in resting enzyme contains an extremely high C(epsilon 1)-H proton chemical shift of 9.30 parts per million (ppm) owing to a conserved C(epsilon 1)-H(.)O=C H-bond from the active site imidazole to a backbone carbonyl group, which is found in all known serine proteases representing all four superfamilies. Only His 226, and His 64 at high pH, exist as the rare N(delta1)-H tautomer, exhibiting (13)C(delta1) chemical shifts approximately 9 ppm higher than those for N(epsilon 2)-H tautomers. His 64 in the PMSF complex, unlike that in the resting enzyme, is highly mobile in its low pH form, as shown by (15)N-(1)H NOE effects, and titrates with rapid proton exchange kinetics linked to a pK(a) value of 7.47 +/- 0.02.     

Dynamics of β-CH and β-CH2 Groups of Amino Acid Side Chains in Proteins

The dynamics of amino acid side chains of uniformly 13C/15N-enriched ribonuclease T1 (RNase T1) have been investigated. Heteronuclear longitudinal relaxation rates, 1H/13C NOEs, and transverse cross-correlated cross-relaxation rates between the Sx and the SxIz1Iz2 operators (SIIS cross relaxation) [Ernst and Ernst (1994) J. Magn. Reson., A110, 202-213] have been determined in this study. New pulse sequences for measuring the longitudinal relaxation time and the heteronuclear NOE of aliphatic side chain carbon nuclei were developed using the CCONH type of magnetization transfer and 1HN detection. In addition, an improved pulse sequence for the determination of the SIIS cross relaxation is presented. For the analysis of the relaxation rates, the model of restricted rotational diffusion around the 1 dihedral angle has been applied [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159-7165]. These techniques were used in order to describe the side chain dynamics of the small globular protein RNase T1 (104 amino acids, MW about 11 kDa). Qualitative values of microdynamical parameters were obtained for 73 out of 85 amino acid side chains (glycine and alanine residues excepted) whereas more quantitative values were derived for 67 -CH and -CH2 groups.     

Probing protein side chain dynamics via 13C NMR relaxation

Protein side chain dynamics is associated with protein stability, folding, and intermolecular interactions. Detailed dynamics information is crucial for the understanding of protein function and biochemical and biophysical properties, which can be obtained using NMR relaxation techniques. In this review, (13)C relaxation of methine, methylene and methyl groups with and without (1)H decoupling are described briefly for a better understanding of how spin relaxation is associated with motional (dynamics) parameters. Developments in the measurement and interpretation of (13)C auto-relaxation and cross-correlated relaxation data are presented too. Finally, recent progress in the use of (13)C relaxation to probe the dynamics of protein side chains is detailed mainly for the dynamics of non-deuterated proteins on picoseconds-nanosecond timescales.     

Insight into the binding of antifreeze proteins to ice surfaces via 13C spin lattice relaxation solid-state NMR

The primary sequences of type I antifreeze proteins (AFPs) are Ala rich and contain three 11-residue repeat units beginning with threonine residues. Their secondary structures consist of alpha-helices. Previous activity study of side-chain mutated AFPs suggests that the ice-binding side of type I AFPs comprises the Thr side chains and the conserved i + 4 and i + 8 Ala residues, where i indicates the positions of the Thrs. To find structural evidence for the AFP's ice-binding side, a variable-temperature dependent (13)C spin lattice relaxation solid-state NMR experiment was carried out for two Ala side chain (13)C labeled HPLC6 isoforms of the type I AFPs each frozen in H(2)O and D(2)O, respectively. The first one was labeled on the equivalent 17th and 21st Ala side chains (i + 4, 8), and the second one on the equivalent 8th, 19th, and 30th Ala side chains (i + 6). The two kinds of labels are on the opposite sides of the alpha-helical AFP. A model of Ala methyl group rotation/three-site rotational jump combined with water molecular reorientation was tested to probe the interactions of the methyl groups with the proximate water molecules. Analysis of the T(1) data shows that there could be 10 water molecules closely capping an i + 4 or an i + 8 methyl group within the range of van der Waals interaction, whereas the surrounding water molecules to the i + 6 methyl groups could be looser. This study suggests that the side of the alpha-helical AFP comprising the i + 4 and i + 8 Ala methyl groups could interact with the ice surface in the ice/water interface.     

Histidine side-chain dynamics and protonation monitored by 13C CPMG NMR relaxation dispersion

The use of ¹³C NMR relaxation dispersion experiments to monitor micro-millisecond fluctuations in the protonation states of histidine residues in proteins is investigated. To illustrate the approach, measurements on three specifically ¹³C labeled histidine residues in plastocyanin (PCu) from Anabaena variabilis (A.v.) are presented. Significant Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion is observed for ¹³C^ε1 nuclei in the histidine imidazole rings of A.v. PCu. The chemical shift changes obtained from the CPMG dispersion data are in good agreement with those obtained from the chemical shift titration experiments, and the CPMG derived exchange rates agree with those obtained previously from ¹⁵N backbone relaxation measurements. Compared to measurements of backbone nuclei, ¹³C^ε1 dispersion provides a more direct method to monitor interchanging protonation states or other kinds of conformational changes of histidine side chains or their environment. Advantages and shortcomings of using the ¹³C^ε1 dispersion experiments in combination with chemical shift titration experiments to obtain information on exchange dynamics of the histidine side chains are discussed.     

Conformational exchange of aromatic side chains characterized by L-optimized TROSY-selected 13C CPMG relaxation dispersion

Protein dynamics on the millisecond time scale commonly reflect conformational transitions between distinct functional states. NMR relaxation dispersion experiments have provided important insights into biologically relevant dynamics with site-specific resolution, primarily targeting the protein backbone and methyl-bearing side chains. Aromatic side chains represent attractive probes of protein dynamics because they are over-represented in protein binding interfaces, play critical roles in enzyme catalysis, and form an important part of the core. Here we introduce a method to characterize millisecond conformational exchange of aromatic side chains in selectively (13)C labeled proteins by means of longitudinal- and transverse-relaxation optimized CPMG relaxation dispersion. By monitoring (13)C relaxation in a spin-state selective manner, significant sensitivity enhancement can be achieved in terms of both signal intensity and the relative exchange contribution to transverse relaxation. Further signal enhancement results from optimizing the longitudinal relaxation recovery of the covalently attached (1)H spins. We validated the L-TROSY-CPMG experiment by measuring fast folding-unfolding kinetics of the small protein CspB under native conditions. The determined unfolding rate matches perfectly with previous results from stopped-flow kinetics. The CPMG-derived chemical shift differences between the folded and unfolded states are in excellent agreement with those obtained by urea-dependent chemical shift analysis. The present method enables characterization of conformational exchange involving aromatic side chains and should serve as a valuable complement to methods developed for other types of protein side chains.     

Internal motions of apo-neocarzinostatin as studied by 13C NMR methine relaxation at natural abundance

Summary Dynamics of the backbone and some side chains of apo-neocarzinostatin, a 10.7 kDa carrier protein, have been studied from ¹³C relaxation rates R1, R2 and steady-state ¹³C-{¹H} NOEs, measured at natural abundance. Relaxation data were obtained for 79 nonoverlapping C resonances and for 11 threonine C single resonances. Except for three C relaxation rates, all data were analysed from a simple two-parameter spectral density function using the model-free approach of Lipari and Szabo. The corresponding C–H fragments exhibit fast (_e < 40 ps) restricted libration motions (S²=0.73 to 0.95). Global examination of the microdynamical parameters S² and _e along the amino acid sequence gives no immediate correlation with structural elements. However, different trends for the three loops involved in the binding site are revealed. The -ribbon comprising residues 37 to 47 is spatially restricted, with relatively large _e values in its hairpin region. The other -ribbon (residues 72 to 87) and the large disordered loop ranging between residues 97–107 experience small-amplitude motions on a much faster (picosecond) time scale. The two N-terminal residues, Ala¹ and Ala², and the C-terminal residue Asn¹¹³, exhibit an additional slow motion on a subnanosecond time scale (400–500 ps). Similarly, the relaxation data for eight threonine side-chain C must be interpreted in terms of a three-parameter spectral density function. They exhibit slower motions, on the nanosecond time scale (500–3000 ps). Three threonine (Thr⁶⁵, Thr⁶⁸, Thr⁸¹) side chains do not display a slow component, but an exchange contribution to the observed transverse relaxation rate R2 could not be excluded at these sites. The microdynamical parameters (S², _e and R2_ex) or (S _infslow ^sup2 , S _inffast ^sup2 and _slow) were obtained from a straightforward solution of the equations describing the relaxation data. They were calculated assuming an overall isotropic rotational correlation time _e for the protein of 5.7 ns, determined using standard procedures from R2/R1 ratios. However, it is shown that the product (1–S²)× _e is nearly independent of _e for residues not exhibiting slow motions on the nanosecond time scale. In addition, this parameter very closely follows the heteronuclear NOEs, which therefore could be good indices for local fast motions on the picosecond time scale.     

Structural features of pectic polysaccharides from the skin of Opuntia ficus-indica prickly pear fruits

After removal of the mucilage with water at room temperature, pectic polysaccharides were solubilized from Opuntia ficus-indica fruit skin, by sequential extraction with water at 60 degrees C (WSP) and EDTA solution at 60 degrees C (CSP). Polysaccharides with neutral sugar content of 0.48 and 0.36 mol/mol galacturonic acid residue were obtained, respectively, in the WSP and CSP extracts. These pectic polysaccharides were de-esterified and fractionated by anion-exchange chromatography, yielding for each extract five fractions, which were thereafter purified by size-exclusion chromatography. Two of these purified fractions were characterized by sugar analysis combined with methylation and reduction-methylation analysis. The study was then supported by (1)H and (13)C NMR spectroscopy. The results showed that the water-soluble fraction WSP3 and the EDTA soluble fraction CSP3, consisted of a disaccharide repeating unit -->2)-alpha-l-Rhap-(1-->4)-alpha-d-GalpA-(1--> backbone, with side chains attached to O-4 of the rhamnosyl residues. The side chains contained highly branched alpha-(1-->5)-linked arabinan and short linear beta-(1-->4)-linked galactan.     

Side chain dynamics monitored by 13C-13C cross-relaxation

A method to measure (13)C-(13)C cross-relaxation rates in a fully (13)C labeled protein has been developed that can give information about the mobility of side chains in proteins. The method makes use of the (H)CCH-NOESY pulse sequence and includes a suppression scheme for zero-quantum (ZQ) coherences that allows the extraction of initial rates from NOE buildup curves.The method has been used to measure (13)C-(13)C cross-relaxation rates in the 269-residue serine-protease PB92. We focused on C(alpha)-C(beta) cross-relaxation rates, which could be extracted for 64% of all residues, discarding serine residues because of imperfect ZQ suppression, and methyl (13)C-(13)C cross-relaxation rates, which could be extracted for 47% of the methyl containing C-C pairs. The C(alpha)-C(beta) cross-relaxation rates are on average larger in secondary structure elements as compared to loop regions, in agreement with the expected higher rigidity in these elements. The cross-relaxation rates for methyl containing C-C pairs show a general decrease of rates further into the side chain, indicating more flexibility with increasing separation from the main chain. In the case of leucine residues also long-range C(beta)-C(delta) cross-peaks are observed. Surprisingly, for most of the leucines a cross-peak with only one of the methyl C(delta) carbons is observed, which correlates well with the chi(2) torsion-angle and can be explained by a difference in mobility for the two methyl groups due to an anisotropic side chain motion.     

锌酵母中酵母甘露多糖组分的特征和结构

本文研究从锌酵母中分离出的酵母甘露多糖ＸＰ的特征和结构。ＸＰ经全水解和＾１３ＣＮＭＲ谱显示除甘露糖基外,还有少量Ｌ－鼠李糖基和甲氧基。甲基化分析、过碘酸盐氧化、Ｓｍｉｔｈ降解、乙酰解和部分酸水解显示ＸＰ的主链是１→６连接的甘露糖,侧链是１→２连接的甘露糖。＾１Ｈ及＾１３Ｃ ＮＭＲ谱表明所有糖苷键均为α型,结合元素分析ＸＰ基本是酵母甘露多糖和蛋白质以及锌的络合物。     

Selectively 13C-enriched DNA: Dynamics of the C1′-H1′ vector in d(CGCAAATTTGCG)2

Summary In order to examine the internal dynamic processes of the dodecamer d(CGCAAATTTGCG)₂, the ¹³C-enriched oligonucleotide has been synthesized. The three central thymines were selectively ¹³C-labeled at the C1′ position and their spin-lattice relaxation parameters R(C_Z), R(C_X,Y), R(H_Z→C_Z), R(2H_ZC_Z), R(2H_ZC_X,Y) and R(H _infZ ^supC ) were measured. Density functions were computed for two models of internal motions. Comparisons of the experimental data were made with the spin-lattice relaxation rates rather than with the density functions, whose values were altered by accumulation of the uncertainties of each relaxation rate measurement. The spin-lattice relaxation rates were computed with respect to the motions of the sugar around the C1′-N1 bond. A two-state jump model between the anti- and syn-conformations with P(anti)/P(syn)=91/9 or a restricted rotation model with Δχ=28° and an internal diffusion coefficient of 30×10⁷ s^-1 gave a good fit with the experimental data. Twist, tilt or roll base motions have little effect on ¹³C1′ NMR relaxation. Simulation of spin-relaxation rates with the data obtained at several temperatures between 7 and 32 °C, where the dodecamer is double stranded, shows that the internal motion amplitude is independent of the temperature within this range, as expected for internal motion. Using the strong correlation which exists in a B-DNA structure between the χ and δ angle, we suggest that the change in the glycosidic angle value should be indicative of a sugar puckering between the C1′-exo and C2′-endo conformations.     

Estimates of methyl 13C and 1H CSA values (Δσ) in proteins from cross-correlated spin relaxation

Simple pulse schemes are presented for the measurement of methyl ¹³C and ¹H CSA values from ¹H–¹³C dipole/¹³C CSA and ¹H–¹³C dipole/¹H CSA cross-correlated relaxation. The methodology is applied to protein L and malate synthase G. Average ¹³C CSA values are considerably smaller for Ile than Leu/Val (17 vs 25 ppm) and are in good agreement with previous solid state NMR studies of powders of amino acids and dipeptides and in reasonable agreement with quantum-chemical DFT calculations of methyl carbon CSA values in peptide fragments. Small averaged ¹H CSA values on the order of 1 ppm are measured, consistent with a solid state NMR determination of the methyl group ¹H CSA in dimethylmalonic acid.     

Biosynthesis of benzofuran derivatives in root cultures of Tagetes patula via phenylalanine and 1-deoxy-D-xylulose 5-phosphate

Root cultures of Tagetes patula L. cv. Carmen were grown with a mixture of unlabeled glucose and [U-(13)C(6)]glucose or [1-(13)C(1)]glucose as carbon source. Isoeuparin and (-)-4-hydroxytremetone were isolated by solvent extraction of the cultured tissue, purified by chromatography and analysed by (1)H and (13)C NMR spectroscopy. Amino acids obtained by hydrolysis of protein from the same experiments were used for the reconstruction of the labelling patterns in central metabolic intermediates. These labelling patterns were used for the prediction of isotopolog compositions in the benzofuranone derivatives via different hypothetical pathways. Comparison with the experimentally observed isotopolog distributions showed that the benzenoid ring and the acetoxy group are exclusively or predominantly (>98%) derived from phenylalanine and not from acetyl-CoA via a polyketide-type biosynthesis. The isopropylidene side chain and two carbon atoms of the furan and dihydrofuran moiety, respectively, originate from an isoprenoid building block obtained exclusively or predominantly (>98%) via the deoxyxylulose phosphate pathway. The exomethylene atom of the isopropylidene side chain is biosynthetically equivalent to the (Z)-methyl group of dimethylallyl diphosphate. The data indicate that isoeuparin and (-)-4-hydroxytremetone are assembled from 4-hydroxyacetophenone and dimethylallyl diphosphate via prenyl-substituted 4-hydroxyacetophenone and dihydrobenzofurans as intermediates.     

13C-NMR studies of the membrane structure of enveloped virions (vesicular stomatitis virus).

The mobility of the lipids in the bilayer of the envelope of vesicular stomatitis virus has been probed over its complete space by the biosynthetic incorporation of [N-13CH3]- choline as a probe for the polar head groups and [3-13C]- and [11-13C] oleic acid and [16-13C]- palmitic acid for the hydrophobic region of the bilayer. These precursors were effectively incorporated as established by the concomitant administration of the same precursors in radioactive form. Spin lattice relaxation time measurements (T1) of the 13C enriched segments in complete virus envelope allowed estimation of their mobility. The mobility of the polar head groups is restricted, probably due to ionic interactions with neighbouring acidic phospholipids (phosphatidylserine) and/or acidic side chains of the glycoprotein (G-protein). The rigidity of the hydrophobic part of the bilayer is due to the high cholesterol content and interaction with the immersing polypeptide chains of the G- and possibly M-protein. The rigidity is limited to a depth of about 15 A ranging from the inner and outer surface, whereas the inner core of the bilayer is fluid. Tryptic cleavage of the hydrophilic part of the G-protein allows the lipophilic immersing polypeptide fragment to enter further the bilayer which then reduces the fluidity of the hydrocarbon chains in the core region by lipid-protein interactions.     

Why does insect antifreeze protein from Tenebrio molitor produce pyramidal ice crystallites?

The antifreeze protein (AFP) reduces the growth rates of the ice crystal facets. In that process the ice morphology undergoes a modification. An AFP-induced surface pinning mechanism, through matching of periodic bond chains in two dimensions, enables two-dimensional regular ice-binding surfaces (IBSs) of the insect AFPs to engage a certain class of ice surfaces, called primary surfaces. They are kinetically stable surfaces with unambiguous and predetermined orientations. In this work, the orientations and molecular compositions of the primary ice surfaces that undergo growth rate reduction by the insect AFPs are obtained from first principles. Besides the basal face and primary prism, the ice surfaces engaged by insect AFPs include the specific ice pyramids produced by the insect AFP Tenebrio molitor (TmAFP). TmAFP-induced pyramids differ fundamentally from the ice pyramids produced by fish AFPs and antifreeze protein glycoproteins (AFPGs) as regards the ice surface configurations and the mode of interaction with the protein IBS. The molecular compositions of the TmAFP-induced pyramids are strongly bonded in two dimensions and have the constant face indices (101). In contrast, the molecular composition of the ice pyramids produced by fish AFPs and AFPGs are strongly bonded in only one direction and have variable face indices (h 0 l), none of which equal (101). The thus far puzzling behavior of the TmAFP in producing pyramidal crystallites is fully explained in agreement with experiment.