Protein structure and function at low temperatures

Proteins represent the major components in the living cell that provide the whole repertoire of constituents of cellular organization and metabolism. In the process of evolution, adaptation to extreme conditions mainly referred to temperature, pH and low water activity. With respect to life at low temperatures, effects on protein structure, protein stability and protein folding need consideration. The sequences and topologies of proteins from psychrophilic, mesophilic and thermophilic organisms are found to be highly homologous. Commonly, adaptive changes refer to multiple alterations of the amino acid sequence, which presently cannot be correlated with specific changes of structure and stability; so far it has not been possible to attribute specific increments in the free energy of stabilization to well-defined amino-acid exchanges in an unambiguous way. The stability of proteins is limited at high and low temperatures. Their expression and self-organization may be accomplished under conditions strongly deviating from optimum growth conditions. Molecular adaptation to extremes of temperature seems to be accompanied by a flattening of the temperature profile of the free energy of stabilization. In principle, the free energy of stabilization of proteins is small compared to the total molecular energy. As a consequence, molecular adaptation to extremes of physical conditions only requires marginal alterations of the intermolecular interactions and packing density. Careful statistical and structural analyses indicate that altering the number of ion pairs and hydrophobic interactions allows the flexibility of proteins to be adjusted so that full catalytic function is maintained at varying temperatures.     

Glycerol effects on protein flexibility: a tryptophan phosphorescence study.

In exploring the dynamic properties of protein structure, numerous studies have focussed on the dependence of structural fluctuations on solvent viscosity, but the emerging picture is still not well defined. Exploiting the sensitivity of the phosphorescence lifetime of tryptophan to the viscosity of its environment we have used the delayed emission as an intrinsic probe of protein flexibility and investigated the effects of glycerol as a viscogenic cosolvent. The phosphorescence lifetime of alcohol dehydrogenase, alkaline phosphatase, apoazurin and RNase T1, as a function of glycerol concentration was studied at various temperatures. Flexibility data, which refer to rather rigid sites of the globular structures, point out that, for some concentration ranges glycerol, effects on the rate of structural fluctuations of alcohol dehydrogenase and RNase T1 do not obey Kramers' a power law on solvent viscosity and emphasize that cosolvent-induced structural changes can be important, even for inner cores of the macromolecule. When the data is analyzed in terms of Kramers' model, for the temperature range 0-30 degrees C one derives frictional coefficients that are relatively large (0.6-0.7) for RNase T1, where the probe is in a flexible region near the surface of the macromolecule and much smaller, less than 0.2, for the rigid sites of the other proteins. For the latter sites the frictional coefficient rises sharply between 40 and 60 degrees C, and its value correlates weakly with molecular parameters such as the depth of burial or the rigidity of a particular site. For RNase T1, coupling to solvent viscosity increases at subzero temperatures, with the coefficient becoming as large as 1 at -20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability and stabilization of globular proteins in solution

Proteins are multifunctional: their amino acid sequences simultaneously determine folding, function and turnover. Correspondingly, evolution selected for compromises between rigidity (stability) and flexibility (folding/function/degradation), to the result that generally the free energy of stabilization of globular proteins in solution is the equivalent to only a few weak intermolecular interactions. Additional increments may come from extrinsic factors such as ligands or specific compatible solutes. Apart from the enthalpic effects, entropy may play a role by reducing the flexibility (cystine bridges, increased proline content), or by water release from residues buried upon folding and association. Additional quaternary interactions and closer packing are typical characteristics of proteins from thermophiles. In halophiles, protein stability and function are maintained by increased ion binding and glutamic acid content, both allowing the protein inventory to compete for water at high salt. Acidophiles and alkalophiles show neutral intracellular pH; proteins facing the outside extremes of pH possess anomalously high contents in ionizable amino acids. Global comparisons of the amino acid compositions and sequences of proteins from mesophiles and extremophiles did not result in general rules of protein stabilization, even after including complete genome sequences into the search. Obviously, proteins are individuals that optimize internal packing and external solvent interactions by very different mechanisms, each protein in its own way. Strategies deduced from specific ultrastable proteins allow stabilizing point mutations to be predicted.     

ENVIRON: a software package to compare protein three-dimensional structures with homologous sequences using local structural motifs

This work presents a method to compare local clusters of interactingresidues as observed in a known three-dimensional protein structurewith corresponding clusters inferred from homologous proteinsequences, assuming conserved protein folding. For this purposethe local environment of a selected residue in a known proteinstructure is defined as the ensemble of amino acids in contactwith it in the folded state. Using a multiple sequence alignmentto identify corresponding residues in homologous proteins, adetailed comparison can be performed between the local environmentof a selected amino acid in the template protein structure andthe expected local environments at the sets of equivalent residues,derived from the aligned protein sequences. The comparison makesit possible to detect conserved local features such as hydrogenbonding or complementarity in residue substitution. A globalmeasure of environmental similarity is also defined, to searchfor conserved amino acid clusters subject to functional or structural constraints. The proposed approach is useful for investigatingprotein function as well as for site-directed mutagenesis experiments,where appropriate amino acid substitutions can be suggestedby observing naturally occurring protein variants.     

Solution structure and backbone dynamics of the K18G/R82E Alicyclobacillus acidocaldarius thioredoxin mutant: a molecular analysis of its reduced thermal stability

No general strategy for thermostability has been yet established, because the extra stability of thermophiles appears to be the sum of different cumulative stabilizing interactions. In addition, the increase of conformational rigidity observed in many thermophilic proteins, which in some cases disappears when mesophilic and thermophilic proteins are compared at their respective physiological temperatures, suggests that evolutionary adaptation tends to maintain corresponding states with respect to conformational flexibility. In this study, we accomplished a structural analysis of the K18G/R82E Alicyclobacillus acidocaldarius thioredoxin (BacTrx) mutant, which has reduced heat resistance with respect to the thermostable wild-type. Furthermore, we have also achieved a detailed study, carried out at 25, 45, and 65 degrees C, of the backbone dynamics of both the BacTrx and its K18G/R82E mutant. Our findings clearly indicate that the insertion of the two mutations causes a loss of energetically favorable long-range interactions and renders the secondary structure elements of the double mutants more similar to those of the mesophilic Escherichia coli thioredoxin. Moreover, protein dynamics analysis shows that at room temperature the BacTrx, as well as the double mutant, are globally as rigid as the mesophilic thioredoxins; differently, at 65 degrees C, which is in the optimal growth temperature range of A. acidocaldarius, the wild-type retains its rigidity while the double mutant is characterized by a large increase of the amplitude of the internal motions. Finally, our research interestingly shows that fast motions on the pico- to nanosecond time scale are not detrimental to protein stability and provide an entropic stabilization of the native state. This study further confirms that protein thermostability is reached through diverse stabilizing interactions, which have the key role to maintain the structural folding stable and functional at the working temperature.     

Compositional and Structural Features Related to Thermal Stability in the Archaea SRP19 and SRP54 Signal Recognition Particle Proteins

To survive at high temperature, thermophile organisms must adapt their biomolecules. In both nucleic acids and proteins, this adaptation involves a vast array of compositional and structural modifications. The archaea stand out as the only group of organisms that have species capable of growing at temperatures ranging from 0 to 110°C. In this study, we have used the archaea genome datasets to identify molecular trends related to thermal adaptation in the protein components (SRP19 and SRP54) of the signal recognition particle (SRP). Using comparative genomics and secondary structure homology modeling we have detected significant differences in the amino acids composition and distribution between the SRP proteins of thermophile and mesophile archaea. These include: a significant increase in the thermophile SRP proteins of the frequency of charged amino acids able to participate in electrostatic interactions which contribute to stabilize proteins; decreased content of both thermolabile and small/tiny amino acids which usually contribute to protein flexibility; and a significant increase in aliphatic and aromatic amino acids providing good covering and masking to produce hydrophobic pockets involved in stabilizing protein structure. Moreover, a detailed analysis of the four structural and functional domains of the SRP54 indicates a particularly robust correlation between the compositional properties of the M domain and the optimal growth temperature (OGT) of the archaea. The analysis of the bacterial SRP54(Ffh) shows similar adaptations to the OGT. Thus, natural selection has adapted the SRP proteins to the OGT of the archaea and bacteria species by modifying both, their amino acids composition and distribution.     

Comparison of diverse protein sequences of the nuclear-encoded subunits of cytochrome C oxidase suggests conservation of structure underlies evolving functional sites

Interspecific comparisons of protein sequences can reveal regions of evolutionary conservation that are under purifying selection because of functional constraints. Interpreting these constraints requires combining evolutionary information with structural, biochemical, and physiological data to understand the biological function of conserved regions. We take this integrative approach to investigate the evolution and function of the nuclear-encoded subunits of cytochrome c oxidase (COX). We find that the nuclear-encoded subunits evolved subsequent to the origin of mitochondria and the subunit composition of the holoenzyme varies across diverse taxa that include animals, yeasts, and plants. By mapping conserved amino acids onto the crystal structure of bovine COX, we show that conserved residues are structurally organized into functional domains. These domains correspond to some known functional sites as well as to other uncharacterized regions. We find that amino acids that are important for structural stability are conserved at frequencies higher than expected within each taxon, and groups of conserved residues cluster together at distances of less than 5 A more frequently than do randomly selected residues. We, therefore, suggest that selection is acting to maintain the structural foundation of COX across taxa, whereas active sites vary or coevolve within lineages.     

Protein rigidity and thermophilic adaptation

We probe the hypothesis of corresponding states, according to which homologues from mesophilic and thermophilic organisms are in corresponding states of similar rigidity and flexibility at their respective optimal temperatures. For this, the local distribution of flexible and rigid regions in 19 pairs of homologous proteins from meso- and thermophilic organisms is analyzed and related to activity characteristics of the enzymes by constraint network analysis (CNA). Two pairs of enzymes are considered in more detail: 3-isopropylmalate dehydrogenase and thermolysin-like protease. By comparing microscopic stability features of homologues with the help of stability maps, introduced for the first time, we show that adaptive mutations in enzymes from thermophilic organisms maintain the balance between overall rigidity, important for thermostability, and local flexibility, important for activity, at the appropriate working temperature. Thermophilic adaptation in general leads to an increase of structural rigidity but conserves the distribution of functionally important flexible regions between homologues. This finding provides direct evidence for the hypothesis of corresponding states. CNA thereby implicitly captures and unifies many different mechanisms that contribute to increased thermostability and to activity at high temperatures. This allows to qualitatively relate changes in the flexibility of active site regions, induced either by a temperature change or by the introduction of mutations, to experimentally observed losses of the enzyme function. As for applications, the results demonstrate that exploiting the principle of corresponding states not only allows for successful thermostability optimization but also for guiding experiments in order to improve enzyme activity in protein engineering.     

Structures and analysis of highly homologous psychrophilic, mesophilic, and thermophilic adenylate kinases

The crystal structures of adenylate kinases from the psychrophile Bacillus globisporus and the mesophile Bacillus subtilis have been solved and compared with that from the thermophile Bacillus stearothermophilus. This is the first example we know of where a trio of protein structures has been solved that have the same number of amino acids and a high level of identity (66-74%) and yet come from organisms with different operating temperatures. The enzymes were characterized for their own thermal denaturation and inactivation, and they exhibited the same temperature preferences as their source organisms. The structures of the three highly homologous, dynamic proteins with different temperature-activity profiles provide an opportunity to explore a molecular mechanism of cold and heat adaptation. Their analysis suggests that the maintenance of the balance between stability and flexibility is crucial for proteins to function at their environmental temperatures, and it is achieved by the modification of intramolecular interactions in the process of temperature adaptation.     

Manipulation of the napin primary structure alters its packaging and deposition in transgenic tobacco (Nicotiana tabacum L.) seeds

Napin is a 2S storage protein found in the seeds of oilseed rape (Brassica napus L.) and related species. Using protein structural prediction programs we have identified a region in the napin protein sequence which forms a `hydrophilic loop' composed of amino acid residues located at the protein surface. Targeting this region, we have constructed two napin chimeric genes containing the coding sequence for the peptide hormone leucine-enkephalin as a topological marker. One version has a single enkephalin sequence of 11 amino acids including linkers and the second contains a tandem repeat of this peptide comprising 22 amino acids, inserted into the napin large subunit. The inserted peptide sequences alter the balance of hydrophilic to hydrophobic amino acids and introduce flexibility into this region of the polypeptide chain. The chimeric genes have been expressed in tobacco plants under the control of the seed-specific napA gene promoter. Analyses indicate that the engineered napin proteins are expressed, transported, post-translationally modified and deposited inside the protein bodies of the transgenic seeds demonstrating that the altered napin proteins behave in a similar fashion to the authentic napin protein. Detailed immunolocalisation studies indicate that the insertion of the peptide sequences has a significant effect on the distribution of the napin proteins within the tobacco seed protein bodies.     

Stability and solubility of proteins from extremophiles

Charges are important for hyperthermophile protein structure and function. However, the number of charges and their predicted contributions to folded state stability are not correlated, implying that more charge does not imply greater stability. The charge properties that distinguish hyperthermophile proteins also differentiate psychrophile proteins from mesophile proteins, but in the opposite direction and to a smaller extent. We conclude that charge number relates to solubility, whereas protein stability is determined by charge location. Most other structural properties are poorly separated over the ambient temperature range, apart from the burial of certain amino acids. Of particular interest are large non-polar sidechains that tend to increased exposure in proteins evolved to function at higher temperatures. Looking at tryptophan in more detail, this increase is often located close to the termini of secondary structure elements, and is discussed in terms of a novel potential role in protein thermostabilisation.     

Thinking outside the crystal: complementary approaches for examining transporter conformational change

As the number of high-resolution structures of membrane proteins continues to rise, so has the necessity for techniques to link this structural information to protein function. In the case of transporters, function is achieved via coupling of conformational changes to substrate binding and release. Static structural data alone cannot convey information on these protein movements, but it can provide a high-resolution foundation on which to interpret lower resolution data obtained by complementary approaches. Here, we review selected biochemical and spectroscopic methods for assessing transporter conformational change. In addition to more traditional techniques, we present 1?F-NMR as an attractive method for characterizing conformational change in transporters of known structure. Using biosynthetic labeling, multiple, non-perturbing fluorine-labeled amino acids can be incorporated throughout a protein to serve as reporters of conformational change. Such flexibility in labeling allows characterization of movement in protein regions that may not be accessible via other methods.     

Amino acid pools in CHL V79 cells during induction of thermotolerance: reduction in free intracellular glutamine

The amino acid pools in Chinese hamster lung V79 cells were measured as a function of time during hyperthermic exposure at 40.5 degrees and 45.0 degrees C. Sixteen of the 20 protein amino acids were present in sufficient quantity to measure accurately. The total amino acid pool and all individual amino acids, except glutamine, remained relatively constant for at least 90 min at 40.5 degrees C and for 30 min at 45 degrees C. The glutamine pool decreased rapidly to 20% of its control value within 30 min at 40.5 degrees C with a T1/2 = 15 min. At 45 degrees C, the decrease was 36%. Thermotolerance developed at 40.5 degrees C with a T1/2 = 30 min; thus, glutamine depletion preceeds the development of thermotolerance. The depletion of glutamine is probably due to increased metabolism and oxidation of glutamine through the TCA cycle at hyperthermic temperatures. Glutamine, as is true for other amino acids, was shown to protect proteins from thermal inactivation and V79 cells from hyperthermic killing when added in excess (4-10 mM) to the medium during heat stress. However, the stability of the total amino acid pool during the development of thermotolerance indicates that resistance to heat does not result from the accumulation of amino acids which then protect against thermal damage. The effects of the large decrease in the glutamine pool are unknown, although glutamine depletion may act as a signal for part of the heat shock response.     

Mechanics of proteins with a focus on atomic force microscopy

The capacity of proteins to function relies on a balance between molecular stability to maintain their folded state and structural flexibility allowing conformational changes related to biological function. Among many others, four different examples can be chosen. The giant protein titin is stretched and can unfold during muscle contraction providing passive elasticity to muscle tissue; myoglobin adsorbs and releases oxygen molecules thank to conformational changes in its structure; the outer membrane protein G (OmpG) is a bacterial porin with a long and flexible loop that modulates gating; and the proton pump bacteriorhodopsin adapts its cytosolic half to allow proton pumping. All these conformational changes triggered either by chemical or by physical cues, require mechanical flexibility or elasticity of certain protein domains. While the methods to determine protein structure, X-ray crystallography above all, have been dramatically improved over the last decades, the number of tools that directly measure the mechanical flexibility of proteins and protein domains is still limited. In this tutorial, after a brief introduction to protein structure, we present some of the available techniques to estimate protein flexibility, then focusing on atomic force microscopy (AFM). We describe the principles of the technique and its various imaging and force spectroscopy modes of operation that allow probing the elasticity of proteins, protein domains and their surrounding environment.     

Cloning and Characterization of a Bacterial Cell-Bound Type B Carboxylesterase from Bacillus sp. BP-7

A clone producing halos on tributyrin plates was isolated from a genomic library of Bacillus sp. BP-7. The insert contained an open reading frame that coded for a protein of 487 amino acids with homology to carboxylesterases. The cloned enzyme showed clear preference for esters of short-chain fatty acids, being classified as an esterase. Maximum activity was found at 45 degrees C and pH 7.5. The enzyme displayed stability in the pH range from 6 to 9.5, and at temperatures from 4 degrees to 45 degrees C. Zymogram analysis of the protein revealed a molecular mass of 53 kDa and a pI of 5.1. The enzyme showed homology to members of the bacterial subclass of type B carboxylesterases, a set of proteins potentially useful for biotechnological applications.     

Thinking outside the crystal: Complementary approaches for examining transporter mechanism

As the number of high-resolution structures of membrane proteins continues to rise, so has the necessity for techniques to link this structural information to protein function. In the case of transporters, function is achieved via coupling of conformational changes to substrate binding and release. Static structural data alone cannot convey information on these protein movements, but it can provide a high-resolution foundation on which to interpret lower resolution data obtained by complementary approaches. Here, we review selected biochemical and spectroscopic methods for assessing transporter conformational change. In addition to more traditional techniques, we present ¹⁹F-NMR as an attractive method for characterizing conformational change in transporters of known structure. Using biosynthetic labeling, multiple, non-perturbing fluorine-labeled amino acids can be incorporated throughout a protein to serve as reporters of conformational change. Such flexibility in labeling allows characterization of movement in protein regions that may not be accessible via other methods.     

Protein motions visualized by femtosecond time-resolved crystallography: The case of photosensory vs photosynthetic proteins

Proteins are dynamic objects and undergo conformational changes when functioning. These changes range from interconversion between states in equilibrium to ultrafast and coherent structural motions within one perturbed state. Time-resolved serial femtosecond crystallography at free-electron X-ray lasers can unravel structural changes with atomic resolution and down to femtosecond time scales. In this review, we summarize recent advances on detecting structural changes for phytochrome photosensor proteins and a bacterial photosynthetic reaction center. In the phytochrome structural changes are extensive and involve major rearrangements of many amino acids and water molecules, accompanying the regulation of its biochemical activity, whereas in the photosynthetic reaction center protein the structural changes are smaller, more localized, and are optimized to facilitate electron transfer along the chromophores. The detected structural motions underpin the proteins’ function, providing a showcase for the importance of detecting ultrafast protein structural dynamics.     

Relationship of protein flexibility to thermostability

Thermostability of proteins arises from the simultaneous effect of several forces, which in fact lead to decreased flexibility of the polypeptide chain. This is verified by flexibility indices, which are derived from normalized B-values of individual amino acids in several refined three-dimensional structures. Flexibility indices show that overall flexibility is reduced when thermostability is increased. Protein molecules require both flexibility and rigidity to function, but the higher the temperature optimum and stability the more rigid is the structure needed to compensate for increased thermal fluctuations. Flexibilities of proteins performing the same catalytic activity seem to be about the same at their temperature optima, but the more rigid thermostable proteins reach the flexibility of thermolabile proteins at higher temperatures. In several proteins such as allosteric enzymes, some local sites of flexibility are highly conserved. The relevance of reduced flexibility to overall stability of proteins is also discussed. Flexibility indices and profiles can be used in the design of more stable proteins by site-directed mutagenesis.     

Genetic analysis reveals domain interactions of Arabidopsis Hsp100/ClpB and cooperation with the small heat shock protein chaperone system

We have defined amino acids important for function of the Arabidopsis thaliana Hsp100/ClpB chaperone (AtHsp101) in acquired thermotolerance by isolating recessive, loss-of-function mutations and a novel semidominant, gain-of-function allele [hot1-4 (A499T)]. The hot1-4 allele is unusual in that it not only fails to develop thermotolerance to 45 degrees C after acclimation at 38 degrees C, but also is sensitive to 38 degrees C, which is a permissive temperature for wild-type and loss-of-function mutants. hot1-4 lies between nucleotide binding domain 1 (NBD1) and NBD2 in a coiled-coil domain that is characteristic of the Hsp100/ClpB proteins. We then isolated two classes of intragenic suppressor mutations of hot1-4: loss-of-function mutations (Class 1) that eliminated the 38 degrees C sensitivity, but did not restore thermotolerance function to hot1-4, and Class 2 suppressors that restored acquired thermotolerance function to hot1-4. Location of the hot1-4 Class 2 suppressors supports a functional link between the coiled-coil domain and both NBD1 and the axial channel of the Hsp100/ClpB hexamer. In addition, the strongest Class 2 suppressors restored solubility of aggregated small heat shock proteins (sHsps) after heat stress, revealing genetic interaction of the Hsp100/ClpB and sHsp chaperone systems. These results also demonstrate that quantitative phenotypes can be used for in vivo genetic dissection of protein mechanism in Arabidopsis.     

Adaptation of model proteins from cold to hot environments involves continuous and small adjustments of average parameters related to amino acid composition

The growth temperature adaptation of six model proteins has been studied in 42 microorganisms belonging to eubacterial and archaeal kingdoms, covering optimum growth temperatures from 7 to 103 degrees C. The selected proteins include three elongation factors involved in translation, the enzymes glyceraldehyde-3-phosphate dehydrogenase and superoxide dismutase, the cell division protein FtsZ. The common strategy of protein adaptation from cold to hot environments implies the occurrence of small changes in the amino acid composition, without altering the overall structure of the macromolecule. These continuous adjustments were investigated through parameters related to the amino acid composition of each protein. The average value per residue of mass, volume and accessible surface area allowed an evaluation of the usage of bulky residues, whereas the average hydrophobicity reflected that of hydrophobic residues. The specific proportion of bulky and hydrophobic residues in each protein almost linearly increased with the temperature of the host microorganism. This finding agrees with the structural and functional properties exhibited by proteins in differently adapted sources, thus explaining the great compactness or the high flexibility exhibited by (hyper)thermophilic or psychrophilic proteins, respectively. Indeed, heat-adapted proteins incline toward the usage of heavier-size and more hydrophobic residues with respect to mesophiles, whereas the cold-adapted macromolecules show the opposite behavior with a certain preference for smaller-size and less hydrophobic residues. An investigation on the different increase of bulky residues along with the growth temperature observed in the six model proteins suggests the relevance of the possible different role and/or structure organization played by protein domains. The significance of the linear correlations between growth temperature and parameters related to the amino acid composition improved when the analysis was collectively carried out on all model proteins.