The parallel helices of the intermediate filaments of alpha-keratin

Recent Fourier transform infrared spectroscopy (FTIR) with attenuated total reflection technique (ATR) has been applied to alpha-keratin fibers (horse-hair) extended in water both at 21 and 95 degrees C. Infrared absorption bands in the Amide 1 region indicated that at extensions to 40-50% strain in water at 21 degrees C alpha-helices had completely disappeared and parallel beta-sheets were formed [Appl. Spectrosc. 55 (2001) 552]. However, when the hair fibers were extended to the same strain at 95 degrees C in water the result was the formation of anti-parallel beta-sheets. These results suggest that the relatively more stable anti-parallel beta-state [Polymer 10 (1969) 810] is only attained in extended alpha-keratin fibers at elevated temperatures and must result from major molecular rearrangement. It was concluded that the alpha-helices in the intermediate filaments (IFs) of alpha-keratin fibers must be parallel. This is in contrast to the previously accepted orientation of anti-parallel alpha-helices, based primarily on findings of X-ray diffraction studies of the structure of beta-keratin in highly extended fibers [Polymer 10 (1969) 810; Keratins, IL: Thomas Springfield (1972); Nature 316 (1985) 767].     

Heat-induced secondary structure and conformation change of bovine serum albumin investigated by Fourier transform infrared spectroscopy

Fourier transform infrared (FT-IR) spectra were measured for an aqueous solution (pD = 5.40) of defatted monomer bovine serum albumin (BSA) over a temperature range of 25-90 degrees C to investigate temperature-induced secondary structure and conformation changes. The curve fitting method combined with the Fourier self-deconvolution technique allowed us to explore details of the secondary structure and conformation changes in defatted BSA. Particularly striking in the FT-IR spectra was an observation of the formation of an irreversible intermolecular beta-sheet of BSA on heating above 70 degrees C. A band at 1630 cm(-1) in the spectra was assigned to short-segment chains connecting alpha-helical segments. The transition temperature for the short-segment chains connecting alpha-helical segments is lower by 17-18 degrees C, when compared to those of the alpha-helix, turn, and intermolecular beta-sheet structures of BSA, suggesting that the alpha-helix and turn structures of BSA are cooperatively denatured on heating. Moreover, the results give an important feature in heat-induced denaturation of BSA that the conformation changes occur twice around both 57 and 75 degrees C. The appearance of two peaks is interpreted by the collapse of the N-terminal BSA domain due to the crevice in the vicinity between domains I and II at low-temperature transition and by the change in cooperative unit composed of the other two BSA domains at high-temperature transition.     

Conformational studies on a peptide fragment representing the RNA-binding N-terminus of a viral coat protein using circular dichroism and NMR spectroscopy

Conformational studies were performed on a synthetic pentacosapeptide representing the RNA-binding N-terminal region of the coat protein of cowpea chlorotic mottle virus. The effects of ionic strength, addition of (oligo)phosphates and temperature on the conformation of this highly positively charged peptide containing six arginines and three lysines were studied. CD experiments show that the peptide has 15-18% alpha-helical conformation and about 80% random-coil conformation in the absence of inorganic salt at 25 degrees C, and 20-21% alpha-helical conformation under the same conditions at 10 degrees C. Addition of inorganic salts results in an increase of alpha-helix content, up to 42% in the presence of oligophosphate with an average chain length of 18 phosphates, which was used as an RNA analog. NMR experiments show that the alpha-helix formation starts in the region between Thr9 and Gln12, and is extended in the direction of the C terminus. Relaxation measurements show that binding to oligophosphates of increasing length results in reduced internal mobilities of the positively charged side chains of the arginyl and lysyl residues and of the side chain of Thr9 in the alpha-helical region. The alpha-helix formation in the N-terminal part of this viral coat protein upon binding of phosphate groups to the positively charged side chains is suggested to play an essential role in RNA binding.     

Probing the conformation of a human apolipoprotein C-1 by amino acid substitutions and trimethylamine-N-oxide.

To test, at the level of individual amino acids, the conformation of an exchangeable apolipoprotein in aqueous solution and in the presence of an osmolyte trimethylamine-N-oxide (TMAO), six synthetic peptide analogues of human apolipoprotein C-1 (apoC-1, 57 residues) containing point mutations in the predicted alpha-helical regions were analyzed by circular dichroism (CD). The CD spectra and the melting curves of the monomeric wild-type and plasma apoC-1 in neutral low-salt solutions superimpose, indicating 31 +/- 4% alpha-helical structure at 22 degrees C that melts reversibly with T(m,WT) = 50 +/- 2 degrees C and van't Hoff enthalpy deltaH(v,WT)(Tm) = 18 +/- 2 kcal/mol. G15A substitution leads to an increased alpha-helical content of 42 +/- 4% and an increased T(m,G15A) = 57 +/- 2 degrees C, which corresponds to stabilization by delta deltaG(app) = +0.4 +/- 1.5 kcal/mol. G15P mutant has approximately 20% alpha-helical content at 22 degrees C and unfolds with low cooperativity upon heating to 90 degrees C. R23P and T45P mutants are fully unfolded at 0-90 degrees C. In contrast, Q31P mutation leads to no destabilization or unfolding. Consequently, the R23 and T45 locations are essential for the stability of the cooperative alpha-helical unit in apoC-1 monomer, G15 is peripheral to it, and Q31 is located in a nonhelical linker region. Our results suggest that Pro mutagenesis coupled with CD provides a tool for assigning the secondary structure to protein groups, which should be useful for other self-associating proteins that are not amenable to NMR structural analysis in aqueous solution. TMAO induces a reversible cooperative coil-to-helix transition in apoC-1, with the maximal alpha-helical content reaching 74%. Comparison with the maximal alpha-helical content of 73% observed in lipid-bound apoC-1 suggests that the TMAO-stabilized secondary structure resembles the functional lipid-bound apolipoprotein conformation.     

Structural study of poly(beta-benzyl-L-aspartate) monolayers at air-liquid interfaces.

As normally studied, in the solid state or in solution, poly(beta-benzyl-L-aspartate) (PBLA) differs from the other helical polyamino acids in that its alpha-helical conformation is most stable in the left-handed rather than in the right-handed form. The slightly lower energy per residue for the left-handed form in PBLA is easily perturbed, however. The helical screw sense can be inverted in a polar environment and, upon heating above 100 degrees C, a distorted left-handed helix or omega-helix is irreversibly formed. From external reflectance Fourier transform infrared measurements at the air-water interface, the conformation of PBLA in the monolayer state is directly established for the first time. The infrared frequencies of the amide bands suggest that right-handed alpha-helices are formed on the surface of water immediately after spreading the monolayers and independently of the polypeptide conformational distribution in the spreading solution. The right-handed helical form prevails throughout the slow compression of the Langmuir monolayers to collapsed films. The helical screw sense can be reversed by lowering the polarity of the aqueous phase. In addition, an alternate conformation similar to the omega-helix forms on addition of small amounts of isopropanol to the aqueous subphase, and appears to be an intermediate in the helix-helix transition.     

Side-chains configurations in coiled coils revealed by the 5.15-A meridional reflection on hard alpha-keratin X-ray diffraction patterns

The origin of the 5.15-A meridional reflection on hard alpha-keratin X-ray diffraction patterns is discussed in terms of side-chains conformations. We show it to reveal specific configurations of the side chains which are common to all two-stranded alpha-helical coiled coils. Combining literature data on crystallised coiled coil pieces and molecular dynamics results with our X-ray diffraction pattern simulations, we propose rules for the attribution of chi1 torsion angles for coiled coils involved in fibres whose structure cannot be resolved at atomic resolution: in a (a b c d e f g) heptad repeat, a and d residues, respectively, adopt mean t and g+ configurations, whereas statistical rules are given for the other residues.     

Self-complementary motifs (SCM) in alpha-crystallin small heat shock proteins

Small heat shock proteins (sHsp) have been implicated in many cell processes involving the dynamics of protein-protein interactions. Two unusual sequences containing self-complementary motifs (SCM) have been identified within the conserved alpha-crystallin domain of sHsps. When two SCMs are aligned in an anti-parallel direction (N to C and C to N), the charged or polar residues form either salt bridges or hydrogen bonds while the non-polar residues participate in hydrophobic interactions. When aligned in reverse order, the residues of these motifs in alpha-crystallin subunits form either hydrophobic and/or polar interactions. Homology based molecular modeling of the C-terminal domain of alpha-crystallin subunits using the crystal structure of MjHSP16.5 suggests that SCM1 and 2 participate in stabilizing secondary structure and subunit interactions. Also there is overwhelming evidence that these motifs are important in the chaperone-like activity of alpha-crystallin subunits. These sequences are conserved and appear to be characteristic of the entire sHsp superfamily. Similar motifs are also present in the Hsp70 family and the immunoglobulin superfamily.     

The structural properties of the transmembrane segment of the integral membrane protein phospholamban utilizing (13)C CPMAS, (2)H, and REDOR solid-state NMR spectroscopy

Solid-state NMR spectroscopic techniques were used to investigate the secondary structure of the transmembrane peptide phospholamban (TM-PLB), a sarcoplasmic Ca(2+) regulator. (13)C cross-polarization magic angle spinning spectra of (13)C carbonyl-labeled Leu39 of TM-PLB exhibited two peaks in a pure 1-palmitoyl-2-oleoyl-phosphocholine (POPC) bilayer, each due to a different structural conformation of phospholamban as characterized by the corresponding (13)C chemical shift. The addition of a negatively charged phospholipid (1-palmitoyl-2-oleoylphosphatidylglycerol (POPG)) to the POPC bilayer stabilized TM-PLB to an alpha-helical conformation as monitored by an enhancement of the alpha-helical carbonyl (13)C resonance in the corresponding NMR spectrum. (13)C-(15)N REDOR solid-state NMR spectroscopic experiments revealed the distance between the (13)C carbonyl carbon of Leu39 and the (15)N amide nitrogen of Leu42 to be 4.2+/-0.2A indicating an alpha-helical conformation of TM-PLB with a slight deviation from an ideal 3.6 amino acid per turn helix. Finally, the quadrupolar splittings of three (2)H labeled leucines (Leu28, Leu39, and Leu51) incorporated in mechanically aligned DOPE/DOPC bilayers yielded an 11 degrees +/-5 degrees tilt of TM-PLB with respect to the bilayer normal. In addition to elucidating valuable TM-PLB secondary structure information, the solid-state NMR spectroscopic data indicates that the type of phospholipids and the water content play a crucial role in the secondary structure and folding of TM-PLB in a phospholipid bilayer.     

Conformation of a group 2 late embryogenesis abundant protein from soybean. Evidence of poly (L-proline)-type II structure

Late embryogenesis abundant (LEA) proteins are members of a large group of hydrophilic, glycine-rich proteins found in plants, algae, fungi, and bacteria known collectively as hydrophilins that are preferentially expressed in response to dehydration or hyperosmotic stress. Group 2 LEA (dehydrins or responsive to abscisic acid) proteins are postulated to stabilize macromolecules against damage by freezing, dehydration, ionic, or osmotic stress. However, the structural and physicochemical properties of group 2 LEA proteins that account for such functions remain unknown. We have analyzed the structural properties of a recombinant form of a soybean (Glycine max) group 2 LEA (rGmDHN1). Differential scanning calorimetry of purified rGmDHN1 demonstrated that the protein does not display a cooperative unfolding transition upon heating. Ultraviolet absorption and circular dichroism spectroscopy revealed that the protein is in a largely hydrated and unstructured conformation in solution. However, ultraviolet absorption and circular dichroism measurements collected at different temperatures showed that the protein exists in equilibrium between two extended conformational states: unordered and left-handed extended helical or poly (L-proline)-type II structures. It is estimated that 27% of the residues of rGmDHN1 adopt or poly (L-proline)-type II-like helical conformation at 12 degrees C. The content of extended helix gradually decreases to 15% as the temperature is increased to 80 degrees C. Studies of the conformation of the protein in solution in the presence of liposomes, trifluoroethanol, and sodium dodecyl sulfate indicated that rGmDHN1 has a very low intrinsic ability to adopt alpha-helical structure and to interact with phospholipid bilayers through amphipathic alpha-helices. The ability of the protein to remain in a highly extended conformation at low temperatures could constitute the basis of the functional role of GmDHN1 in the prevention of freezing, desiccation, ionic, or osmotic stress-related damage to macromolecular structures.     

The secondary structure of echistatin from 1H-NMR, circular-dichroism and Raman spectroscopy.

Detailed biophysical studies have been carried out on echistatin, a member of the disintegrin family of small, cysteine-rich, RGD-containing proteins, isolated from the venom of the saw-scaled viper Echis carinatus. Analysis of circular-dichroism spectra indicates that, at 20 degrees C, echistatin contains no alpha-helix but contains mostly beta-turns and beta-sheet. Two isobestic points are observed as the temperature is raised, the conformational changes associated with that observed between 40 degrees C and 72 degrees C being irreversible. Raman spectra also indicate considerable beta-turn and beta-sheet (20%) structure and an absence of alpha-helical structure. Three of the four disulphide bridges are shown to be in an all-gauche conformation, while the fourth adopts a trans-gauche-gauche conformation. The 1H-NMR spectrum of echistatin has been almost fully assigned. A single conformation was observed at 27 degrees C with the four proline residues adopting only the trans conformation. A large number of backbone amide protons were found to exchange slowly, but no segments of the backbone were found to be in either alpha-helical or beta-sheet conformation. A number of turns could be characterised. An irregular beta-hairpin contains the RGD sequence in a mobile loop at its tip. Two of the four disulphide cross-links have been identified from the NMR spectra. The data presented in this paper will serve to define the structure of echistatin more closely in subsequent studies.     

Unraveling double stranded alpha-helical coiled coils: an x-ray diffraction study on hard alpha-keratin fibers

Transformations of proteins secondary and tertiary structures are generally studied in globular proteins in solution. In fibrous proteins, such as hard alpha-keratin, that contain long and well-defined double stranded alpha-helical coiled coil domains, such study can be directly done on the native fibrous tissue. In order to assess the structural behavior of the coiled coil domains under an axial mechanical stress, wide angle x-ray scattering and small angle x-ray scattering experiments have been carried out on stretched horse hair fibers at relative humidity around 30%. Our observations of the three major axial spacings as a function of the applied macroscopic strain have shown two rates. Up to 4% macroscopic strain the coiled coils were slightly distorted but retained their overall conformation. Above 4% the proportion of coiled coil domains progressively decreased. The main and new result of our study is the observation of the transition from alpha-helical coiled coils to disordered chains instead of the alpha-helical coiled coil to beta-sheet transition that occurs in wet fibers.     

Laser Raman characterization of conformational changes in sarcoplasmic reticulum induced by temperature, Ca2+, and Mg2+

Laser Raman spectroscopy has been used to examine the conformations of the protein and phospholipid components of sarcoplasmic reticulum from rabbit white skeletal muscle. The phospholipid component is shown to have the conformation of fluid, liquid-crystalline lipids, even at 10 degrees C, and no breaks in the lipid conformation are observed in the range of 10-37 degrees C. Protein (predominantly the Ca2+-dependent ATPase) conformation is shown to contain very little beta-sheet structure under all conditions. Absolute content of alpha-helix and random coil or beta-turn could not be determined because of interference in the amide I and III regions. However, the Ca2+-ATPase in sarcoplasmic reticulum appears to undergo a conformational change at 15-18 degrees C which involves removal of a portion of the tryptophan residues from an aqueous environment and an increase in alpha-helical content. This conformation change coincides with a change in slope of Arrhenius plots of ATP hydrolysis activity. Increasing concentrations of Ca2+ and Mg2+ appear to slightly decrease the alpha-helical content of sarcoplasmic reticulum protein.     

Thermally induced alpha-helix to beta-sheet transition in regenerated silk fibers and films

The structure of thin films cast from regenerated solutions of Bombyx mori cocoon silk in hexafluoroisopropyl alcohol (HFIP) was studied by synchrotron X-ray diffraction during heating. A solid-state conformational transition from an alpha-helical structure to the well-known beta-sheet silk II structure occurred at a temperature of approximately 140 degrees C. The transition appeared to be homogeneous, as both phases do not coexist within the resolution of the current study. Modulated differential scanning calorimetry (DSC) of the films showed an endothermic melting peak followed by an exothermic crystallization peak, both occurring near 140 degrees C. Oriented fibers were also produced that displayed this helical molecular conformation. Subsequent heating above the structural transition temperature produced oriented beta-sheet fibers very similar in structure to B. mori cocoon fibers. Heat treatment of silk films at temperatures well below their degradation temperature offers a controllable route to materials with well-defined structures and mechanical behavior.     

Solution conformation of a peptide fragment representing a proposed RNA-binding site of a viral coat protein studied by two-dimensional NMR

The first 25 amino acids of the coat protein of cowpea chlorotic mottle virus are essential for binding the encapsidated RNA. Although an alpha-helical conformation has been predicted for this highly positively charged N-terminal region [Argos, P. (1981) Virology 110, 55-62; Vriend, G., Verduin, B. J. M., & Hemminga, M. A. (1986) J. Mol. Biol. 191, 453-460], no experimental evidence for this conformation has been presented so far. In this study, two-dimensional proton NMR experiments were performed on a chemically synthesized pentacosapeptide containing the first 25 amino acids of this coat protein [Ten Kortenaar, P. B. W., Krüse, J., Hemminga, M. A., & Tesser, G. I. (1986) Int. J. Pept. Protein Res. 27, 401-413]. All resonances could be assigned by a combined use of two-dimensional correlated spectroscopy and nuclear Overhauser enhancement spectroscopy carried out at four different temperatures. Various NMR parameters indicate the presence of a conformational ensemble consisting of helical structures rapidly converting into more extended states. Differences in chemical shifts and nuclear Overhauser effects indicate that lowering the temperature induces a shift of the dynamic equilibrium toward more helical structures. At 10 degrees C, a perceptible fraction of the conformational ensemble consists of structures with an alpha-helical conformation between residues 9 and 17, likely starting with a turnlike structure around Thr9 and Arg10. Both the conformation and the position of this helical region agree well with the secondary structure predictions mentioned above.     

Studies on the conformational properties of CP-10(42-55), the hinge region of CP-10, using circular dichroism and RP-HPLC.

The conformational properties of CP-10(42-55), a peptide corresponding to the hinge region of CP-10, were investigated using circular dichroism spectroscopy and reverse-phase high-performance liquid chromatography (RP-HPLC). The circular dichroism studies indicated that CP-10(42-55) formed considerable secondary structure in the presence of hydrophobic solution environments including 50% acetonitrile, 50% trifluoroethanol and 200 mM sodium dodecyl sulfate, which comprised a mixture of alpha-helix and beta-sheet. The effect of temperature on the conformation of CP-10(42-55) was investigated between 5 and 40 degrees C, with very small changes in the spectra being observed. RP-HPLC was then used to investigate the effect of temperature on the conformation of CP-10(42-55) in the presence of a hydrophobic surface. Using a C18-adsorbent, CP-10(42-55) exhibited a conformational transition at 25 degrees C, which was associated with an increase in the chromatographic contact area and the binding affinity of the peptide for the stationary phase. In addition, near-planar bandbroadening behaviour indicated that conformational species interconverted with rapid rate constants compared with the chromatographic time scale. These results indicated that the conformational change at 25 degrees C in the RP-HPLC system most likely corresponds to the unfolding of an alpha-helical and/or beta-sheet structure to an extended coil structure. Therefore, the strong chemotactic properties of this peptide may be attributed to its ability to form considerable secondary structure in the presence of a hydrophobic environment.     

Infrared characterization of conformational differences in the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine

Fourier transform infrared spectroscopy was used to characterize the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine (1,3-DPPC), a positional isomer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-DPPC). The molecule exists in three distinct phases over the temperature interval 0-70 degrees C. In the low-temperature (LC) phase, the spectra are indicative of acyl chains packed in an orthorhombic subcell, while the carbonyl groups and phosphate ester at the head group show evidence of only partial hydration. The transition from the low-temperature (LC) phase to the intermediate-temperature (L beta) phase at 25 degrees C corresponds to a temperature-induced head-group hydration in which the hydration of the phosphate and carbonyl ester groups results in the reorganization of the hydrocarbon chain-packing subcell from orthorhombic to hexagonal. The transition from the intermediate (L beta) to the high-temperature (L alpha) phase at 37 degrees C is a gel-to-liquid-crystalline phase transition analogous to the 41.5 degrees C transition of 1,2-DPPC. The spectra of the acyl-chain carbonyl groups show evidence of significant differences in molecular conformation at the carbonyl esters in the LC phase. In the L beta and L alpha phases, the carbonyl band contour becomes much more symmetric. However, two components are clearly present in the spectra indicating that the sn-1 and sn-3 carbonyls experience slightly different environments. The observed differences are likely due to a preferred conformation of the phosphocholine group relative to the glycerol backbone. Indications from the infrared spectra of differences in the structure of the C = O groups provide a possible explanation for the selection of the sn-1 chain of 1,3-DPPC by phospholipase A2 on the basis of a preferred head group conformation.     

Direct kinetic evidence for folding via a highly compact, misfolded state.

The 2 S seed storage protein, sunflower albumin 8 (SFA-8), contains an unusually high proportion of hydrophobic residues including 16 methionines (some of which may form a surface hydrophobic patch) in a disulfide cross-linked, alpha-helical structure. Circular dichroism and fluorescence spectroscopy show that SFA-8 is highly stable to denaturation by heating or chaotropic agents, the latter resulting in a reversible two-state unfolding transition. The small m(U) (-4.7 M(-1) at 10 degrees C) and DeltaC(p) (-0.95 kcal mol(-1) K(-1)) values indicate that relatively little nonpolar surface of the protein is exposed during unfolding. Commensurate with the unusual distribution of hydrophobic residues, stopped-flow fluorescence data show that the folding pathway of SFA-8 is highly atypical, in that the initial product of the rapid collapse phase of folding is a compact nonnative state (or collection of nonnative states) that must unfold before acquiring the native conformation. The inhibited folding reaction of SFA-8, in which the misfolded state (m(M) = -0.95 M(-1) at 10 degrees C) is more compact than the transition state for folding (m(T) = -2.5 M(-1) at 10 degrees C), provides direct kinetic evidence for the transient misfolding of a protein.     

Site-directed mutagenesis of the essential arginine of the formate dehydrogenase active centre

Sequence alignment shows that residue Arg 284 (according to the numbering of the residues in formate dehydrogenase, FDH, from the methylotrophic bacterium Pseudomonas sp. 101) is conserved in NAD-dependent FDHs and D-specific 2-hydroxyacid dehydrogenases. Mutation of Arg 284 to glutamine and alanine results in a change of the catalytic, thermodynamic and spectral properties of FDH. In comparison to wild-type, the affinity of the mutants for the substrate (K(formate)m) or the transition state analogue (K(azide)i) decreases and correlates with the ability of the side chain of residue 284 to form H-bonds. In contrast, the affinity for the coenzyme (K(NAD)d or K(NAD)m) is either not affected or increases and correlates inversely with the partial positive charge of the side chain. The temperature dependence of circular dichroism (CD) spectra of the wild-type FDH and its Ala mutant has been studied over the 5-90 degrees C temperature range. Both proteins reveal regions of enhanced conformational mobility at the predenaturing temperatures (40-55 degrees C) associated with a change of enzyme kinetic parameters and a co-operative transition around 55-70 degrees C which is followed by the loss of enzyme activity. CD spectra of the wild-type and mutant proteins were deconvoluted and contributions from various types of secondary structure estimated. It is shown that the co-operative transition at 55-70 degrees C in the FDH protein globule is triggered by a loss of alpha-helical secondary structure. The results confirm the conclusion, from the crystal structures, that Arg 284 is directly involved in substrate binding. In addition this residue seems to exert a major structural role by supporting the catalytic conformation of the enzyme active centre.     

Isolation and characterization of a D-7 LEA protein from pollen that stabilizes glasses in vitro

A heat-soluble protein present in substantial quantities in Typha latifolia pollen was purified to homogeneity. The protein was subjected to cyanogen bromide cleavage, and the peptides produced were separated by HPLC chromatography and sequenced. The two sequences determined were found to be related to the putative D76 LEA protein from Brassica napus seeds and one of them to the D-7 LEA protein from upland cotton. This suggests the pollen protein to be a member of the LEA group III family of proteins. The secondary structure of the protein in solution and in the dry state was investigated using Fourier transform IR spectroscopy. Whereas the protein in solution was highly unordered, being largely in a random coil conformation, the conformation was largely alpha-helical after fast drying. Slow drying reversibly led to both alpha-helical and intermolecular extended beta-sheet structures. When dried in the presence of sucrose, the protein adopted alpha-helical conformation, irrespective of drying rate. The effect of the protein on the stability of sucrose glasses was also investigated. The dehydrated mixture of sucrose and the LEA protein had higher glass transition temperatures and average strength of hydrogen bonding than dehydrated sucrose alone. We suggest that LEA proteins may play a role together with sugars in the formation of a tight hydrogen bonding network in the dehydrating cytoplasm, thus conferring long-term stability.