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.     

DNA determinants important in sequence recognition by Eco RI endonuclease

Alkylation interference and protection methods (Siebenlist, U., and Gilbert, W., (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 122-126) have been utilized to deduce potential DNA contacts involved in specific complex formation between Eco RI endonuclease and its recognition sequence. The endonuclease protected the N7 position (major groove) of the dG and the N3 position (minor groove) of both dA residues within the Eco RI sequence against alkylation by dimethylsulfate, d(GpApApTpTpC), suggesting the presence of poly-peptide in both grooves in the vicinity of affected nitrogens. Results of methylation interference analysis suggest that the N7 of the Eco RI site dG and the N3 of the central dA, d(GpApApTpTpC), are utilized as contacts by the enzyme. The failure to observe interference upon methylation of the 5'-penultimate dA within the sequence implies that the endonuclease does not bond to the N3 of this residue, despite the fact that it is protected against alkylation by the protein. Ethylation interference patterns suggest four major phosphate contacts between endonuclease and each DNA strand. Two of these phosphates are 5'-external to the Eco RI sequence, d(pNpGpApApTpTpC), suggesting involvement of outside phosphates in electrostatic interactions. Moreover, alkylation protection and interference effects on the two DNA strands display perfect 2-fold symmetry. Thus, the endonuclease interacts with a minimum of 10 nucleotide pairs to yield a DNA-protein complex characterized by elements of symmetry. In contrast, specific alkylation effects were not observed in comparable experiments with the endonuclease and a DNA which had been previously methylated by the Eco RI modification enzyme.     

Gaining and losing the thermophilic adaptation in prokaryotes

We studied the evolution of thermophily in prokaryotes using the phylogenetic relationships between 279 bacteria and archaea and their thermophilic amino acid composition signature. Our findings suggest several examples in which the capacity of thermophilic adaptation has been gained or lost over relatively short evolutionary periods throughout the evolution of prokaryotes.     

Genetic determinants of Tibetan high-altitude adaptation

Some highland populations have genetic adaptations that enable their successful existence in a hypoxic environment. Tibetans are protected against many of the harmful responses exhibited by non-adapted populations upon exposure to severe hypoxia, including elevated hemoglobin concentration (i.e., polycythemia). Recent studies have highlighted several genes subject to natural selection in native high-altitude Tibetans. Three of these genes, EPAS1, EGLN1 and PPARA, regulate or are regulated by hypoxia inducible factor, a principal controller of erythropoiesis and other organismal functions. Uncovering the molecular basis of hypoxic adaptation should have implications for understanding hematological and other adaptations involved in hypoxia tolerance. Because the hypoxia response involves a variety of cardiovascular, pulmonary and metabolic functions, this knowledge would improve our understanding of disease mechanisms and could ultimately be translated into targeted therapies for oxygen deprivation, cardiopulmonary and cerebral pathologies, and metabolic disorders such as diabetes and obesity.     

Disentangling genetic and epigenetic determinants of ultrafast adaptation

A major rationale for the advocacy of epigenetically mediated adaptive responses is that they facilitate faster adaptation to environmental challenges. This motivated us to develop a theoretical–experimental framework for disclosing the presence of such adaptation‐speeding mechanisms in an experimental evolution setting circumventing the need for pursuing costly mutation–accumulation experiments. To this end, we exposed clonal populations of budding yeast to a whole range of stressors. By growth phenotyping, we found that almost complete adaptation to arsenic emerged after a few mitotic cell divisions without involving any phenotypic plasticity. Causative mutations were identified by deep sequencing of the arsenic‐adapted populations and reconstructed for validation. Mutation effects on growth phenotypes, and the associated mutational target sizes were quantified and embedded in data‐driven individual‐based evolutionary population models. We found that the experimentally observed homogeneity of adaptation speed and heterogeneity of molecular solutions could only be accounted for if the mutation rate had been near estimates of the basal mutation rate. The ultrafast adaptation could be fully explained by extensive positive pleiotropy such that all beneficial mutations dramatically enhanced multiple fitness components in concert. As our approach can be exploited across a range of model organisms exposed to a variety of environmental challenges, it may be used for determining the importance of epigenetic adaptation‐speeding mechanisms in general.     

Loss of sulfide adaptation ability in a thermophilic Oscillatoria

A spontaneous variant incapable of anoxygenic photosynthesis was derived from a fully competent strain of Oscillatoria amphigramulata which was originally isolated from a high sulfide-containing hot spring of New Zealand. Although the variant (Oa-2) acquired a slight ability to photosynthesize in the presence of 0.3–0.4 mM sulfide, this was only after a 24 h exposure to sulfide and represented oxygenic photosynthesis only. Unlike the parent strain, the incompetent variant never grew in the presence of sulfide >0.05 mM, nor was there any relief of the inhibition by DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] of CO₂ photoincorporation when sulfide was present. The variant strain has retained all of these characteristics over a 4 year period with monthyl transfers in non-sulfide medium. The wild type, under identical conditions, has retained all of its competence with respect to sulfide.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Cold adaptation of the thermophilic enzyme 3-isopropylmalate dehydrogenase

We have performed random mutagenesis coupled with selection to isolate mutant enzymes with high catalytic activities at low temperature from thermophilic 3-isopropylmalate dehydrogenase (IPMDH) originally isolated from Thermus thermophilus. Five cold-adapted mutant IPMDHs with single-amino-acid substitutions were obtained and analyzed. Kinetic analysis revealed that there are two types of cold-adapted mutant IPMDH: k(cat)-improved (improved in k(cat)) and K(m)-improved (improved in k(cat)/K(m)) types. To determine the mechanisms of cold adaptation of these mutants, thermodynamic parameters were estimated and compared with those of the Escherichia coli wild-type IPMDH. The Delta G(m) values for Michaelis intermediate formation of the k(cat)-improved-type enzymes were larger than that of the T. thermophilus wild-type IPMDH and similar to that of the E. coli wild-type IPMDH. The Delta G(m) values of K(m)-improved-type enzymes were smaller than that of the T. thermophilus wild-type IPMDH. Fitting of NAD(+) binding was improved in the K(m)-improved-type enzymes. The two types of cold-adapted mutants employed one of the two strategies of E. coli wild-type IPMDH: relative destabilization of the Michaelis complex in k(cat)-improved-type, and destabilization of the rate-limiting step in K(m)-improved type mutants. Some cold-adapted mutant IPMDHs retained thermostability similar to that of the T. thermophilus wild-type IPMDH.     

DNA sequence determinants for binding of the Escherichia coli catabolite gene activator protein.

The consensus DNA site for binding of the Escherichia coli catabolite gene activator protein (CAP) is 22 base pairs in length and is 2-fold symmetric: 5'-AAATGTGATCTAGATCACATTT-3'. Positions 4 to 8 of each half of the consensus DNA half-site are the most strongly conserved. In this report, we analyze the effects of substitution of DNA base pairs at positions 4 to 8, the effects of substitution of thymine by uracil and by 5-methylcytosine at positions 4, 6, and 8, and the effect of dam methylation of the 5'-GATC-3' sequence at positions 7 to 10. All DNA sites having substitutions of DNA base pairs at positions 4 to 8 exhibit lower affinities for CAP than does the consensus DNA site, consistent with the proposal that the consensus DNA site is the ideal DNA site for CAP. Specificity for T:A at position 4 appears to be determined solely by the thymine 5-methyl group. Specificity for T:A at position 6 and specificity for A:T at position 8 appear to be determined in part, but not solely, by the thymine 5-methyl group. dam methylation has little effect on CAP.DNA complex formation. The thermodynamically defined consensus DNA site spans 28 base pairs. All, or nearly all, DNA determinants required for maximal affinity for CAP and for maximal thermodynamically defined CAP.DNA ion pair formation are contained within a 28-base pair DNA fragment that has the 22-base pair consensus DNA site at its center. The quantitative data in this report provide base-line thermodynamic data required for detailed investigations of amino acid-base pair and amino acid-phosphate contacts in this protein-DNA complex.     

DNA sequence determinants for binding of transformed Ah receptor to a dioxin-responsive enhancer.

We have utilized gel retardation analysis and DNA mutagenesis to examine the specific interaction of transformed guinea pig hepatic cytosolic TCDD.AhR complex with a dioxin-responsive element (DRE). Sequence alignment of the mouse CYPIA1 upstream DREs has identified a common invariant "core" consensus sequence of TNGCGTG flanked by several variable nucleotides. Competitive gel retardation analysis using a series of DRE oligonucleotides containing single or multiple base substitutions has allowed identification of those nucleotides important for TCDD.AhR.DRE complex formation. A putative TCDD.AhR DNA-binding consensus sequence of GCGTGNNA/TNNNC/G has been derived. The four core nucleotides, CGTG, appear to be critical for TCDD-inducible protein-DNA complex formation since their substitution decreased AhR binding affinity by 100-800-fold; the remaining conserved bases are also important, albeit to a lesser degree (3-5-fold). The 5'-ward thymine, present in the invariant core sequence of all the DREs identified to date, appears not to be involved in DNA binding of the AhR. The results obtained here indicate that although the primary interaction of the TCDD.AhR complex with the DRE occurs with the conserved "core" sequence, nucleotides flanking the core also contribute to the specificity of DRE binding.     

On the sequence determinants and flexibility of the kinetoplast DNA fragment with abnormal gel electrophoretic mobilities

A 410 base-pair (bp) Sau3A restriction fragment derived from a Leishmania tarentolae kinetoplast DNA minicircle, which is known to have slower than expected electrophoretic mobilities in polyacrylamide gels, has been cloned in a plasmid and deletions from one end of the cloned segment have been constructed. Analysis of the gel electrophoretic mobility data of a large number of restriction fragments derived from the kinetoplast DNA clone and its deletion subclones has led to the conclusion that two sequences, one in the region bp 100 to 170 and the other bp 190 to 250, both numbered from one end of the 410 bp kinetoplast DNA segment, are important for the abnormal gel electrophoretic behavior of the kinetoplast DNA fragment. One common feature of these sequences is the periodic presence of short runs of A residues (3 to 6 As in each); auto-correlation analysis of these runs of A residues shows a strong harmonic component with a period around 11 bp. These results support and extend the previous analysis of Wu & Crothers (1984). The abnormal electrophoretic behavior is accentuated at low temperature and by the addition of Mg2+ to the electrophoresis buffer; addition of Na+ has the opposite effect. Insertion of sequences derived from the kinetoplast DNA fragment into nicked circular DNA causes no unexpected change in its electrophoretic mobility in agarose gel, suggesting that the 410 bp sequence, or segments of it, has no significant spatial writhe. Abnormal shifts in agarose gel mobilities are observed, however, when certain segments of the kinetoplast DNA are inserted into positively or negatively supercoiled DNA topoisomers. These results are consistent with a bent structure of the kinetoplast DNA in which the bend has zero writhe in its undistorted form but is easily distorted.     

The sequence determinants of cadherin molecules

The sequence and structural analysis of cadherins allow us to find sequence determinants-a few positions in sequences whose residues are characteristic and specific for the structures of a given family. Comparison of the five extracellular domains of classic cadherins showed that they share the same sequence determinants despite only a nonsignificant sequence similarity between the N-terminal domain and other extracellular domains. This allowed us to predict secondary structures and propose three-dimensional structures for these domains that have not been structurally analyzed previously. A new method of assigning a sequence to its proper protein family is suggested: analysis of sequence determinants. The main advantage of this method is that it is not necessary to know all or almost all residues in a sequence as required for other traditional classification tools such as BLAST, FASTA, and HMM. Using the key positions only, that is, residues that serve as the sequence determinants, we found that all members of the classic cadherin family were unequivocally selected from among 80,000 examined proteins. In addition, we proposed a model for the secondary structure of the cytoplasmic domain of cadherins based on the principal relations between sequences and secondary structure multialignments. The patterns of the secondary structure of this domain can serve as the distinguishing characteristics of cadherins.     

Residue determinants and sequence analysis of cold-adapted trypsins

The digestive enzyme trypsin is among the most extensively studied proteins, and its structure has been reported from a large number of organisms. This article focuses on the trypsins from vertebrates adapted to life at low temperatures. Cold-adapted organisms seem to have compensated for the reduced reaction rates at low temperatures by evolving more active and less temperature-stable enzymes. We have analyzed 27 trypsin sequences from a variety of organisms to find unique attributes for the cold-adapted trypsins, comparing trypsins from salmon, Antarctic fish, cod, and pufferfish to other vertebrate trypsins. Both the "cold" and the "warm" active trypsins have about 50 amino acids that are unique and conserved within each class. The main unique features of the cold-adapted trypsins attributable to low-temperature adaptation seem to be (1) reduced hydrophobicity and packing density of the core, mainly because of a lower (Ile + Leu)/(Ile + Leu + Val) ratio, (2) reduced stability of the C-terminal, (3) lack of one warm trypsin conserved proline residue and one proline tyrosine stacking, (4) difference in charge and flexibility of loops extending the binding pocket, and (5) different conformation of the "autolysis" loop that is likely to be involved in substrate binding. Received: January 14, 1999 / Accepted: March 31, 1999     

Protein sequence modules

Conserved protein sequence segments are commonly believed to correspond to functional sites in the protein sequence. A novel approach is proposed to profile the changing degree of conservation along the protein sequence, by evaluating the occurrence frequencies of all short oligopeptides of the given sequence in a large proteome database. Thus, a protein sequence conservation profile can be plotted for every protein. The profile indicates where along the sequences the potential functional (conserved) sites are located. The corresponding oligopeptides belonging to the sites are very frequent across many prokaryotic species. Analysis of a representative set of such profiles reveals a common feature of all examined proteins: they consist of sequence modules represented by the peaks of conservation. Typical size of the modules (peak-to-peak distance) is 25-30 amino acid residues.