Jan Backmann

The crystal structure of triosephosphate isomerase (TIM) from Thermotoga maritima: a comparative thermostability structural analysis of ten different TIM structures.

The molecular mechanisms that evolution has been employing to adapt to environmental temperatures are poorly understood. To gain some further insight into this subject we solved the crystal structure of triosephosphate isomerase (TIM) from the hyperthermophilic bacterium Thermotoga maritima (TmTIM). The enzyme is a tetramer, assembled as a dimer of dimers, suggesting that the tetrameric wild‐type phosphoglycerate kinase PGK‐TIM fusion protein consists of a core of two TIM dimers covalently linked to 4 PGK units. The crystal structure of TmTIM represents the most thermostable TIM presently known in its 3D‐structure. It adds to a series of nine known TIM structures from a wide variety of organisms, spanning the range from psychrophiles to hyperthermophiles. Several properties believed to be involved in the adaptation to different temperatures were calculated and compared for all ten structures. No sequence preferences, correlated with thermal stability, were apparent from the amino acid composition or from the analysis of the loops and secondary structure elements of the ten TIMs. A common feature for both psychrophilic and T. maritima TIM is the large number of salt bridges compared with the number found in mesophilic TIMs. In the two thermophilic TIMs, the highest amount of accessible hydrophobic surface is buried during the folding and assembly process. Proteins 1999;37:441–453. ©1999 Wiley‐Liss, Inc.

Thermally induced hydrogen exchange processes in small proteins as seen by FTIR spectroscopy

Fourier‐transform infrared (FTIR) spectroscopy has been used to study the thermally induced exchange characteristics of those backbone amide protons which persist H‐D exchange at ambient conditions in ribonuclease A, in wild type ribonuclease T1 and some of its variants, and in the histone‐like protein HBsu. The H‐D exchange processes were induced by increasing the thermal energy of the protein solutions in two ways: (i) by linearly increasing the temperature, and (ii) by a temperature jump. To trace the H‐D exchange in the proteins, various infrared absorption bands known to be sensitive to H‐D exchange were used as specific monitors. Characteristic H‐D exchange curves were obtained from which the endpoints (TH/D) of H‐D exchange could be determined. The H‐D exchange curves, the TH/D‐values and the phase transition temperatures Tm were used to estimate the structural flexibility and stability of the given proteins. It is suggested that time‐resolved FTIR spectroscopy can be used to determine global stability parameters of proteins.

Temperature-jump-induced refolding of ribonuclease A: A time-resolved FTIR spectroscopic study

FTIR difference spectroscopy has been used for the first time to investigate the kinetics of secondary structure formation during refolding. The refolding process of ribonuclease A (RNase A) as a model system was induced by applying a temperature‐jump of 60 degrees. The temperature‐jump was triggered by rapidly injecting a small volume of the thermally unfolded protein solution at 80°C into a special cuvette system kept at 20°C. The dead‐time of the injection and the time resolution of the FTIR spectrometer permitted the observation of refolding processes in a time window ranging from 170 ms to several minutes. Specifically, the formation of β‐structures and the disappearance of irregular conformations could be observed in this time interval.

[28] Thermodynamic analysis of hyperthermostable oligomeric proteins

Publisher Summary (Hyper)thermostable proteins differ from their analogs in mesophilic organisms in different intensive parameters, such as packing density, fraction of buried apolar surface, the specific number of hydrogen bonds and of salt bridges, the extension of ion pair networks, and probably the specific amount of β structures. The most important extensive parameter, however, seems to be the number of polypeptide chains per active protein molecule. The higher degree of oligomerization will, of course, influence the above-named intensive parameters. Several proteins in hypothermophilic organisms were found to have a higher degree of oligomerization in comparison to their mesophilic analogs. For studying the thermodynamic stability of oligomeric proteins, several aspects have to be taken into account: (i) the reversibility of unfolding, (ii) the type of the equilibrium between the native and the completely unfolded protein, (iii) the choice of the detection and denaturation method, and (iv) the specific contribution of the association between the monomers to the overall stability.

A correlation between thermal stability and structural features of staphylokinase and selected mutants: a Fourier-transform infrared study

Variants of recombinant staphylokinase (Sak) were investigated by Fourier-transform infrared spectroscopy: Sak (wild type), Sak-M26A, Sak-M26L, and Sak-G34S/R36G/R43H (Sak-B). Estimation of the secondary structure and hydrogen-deuterium exchange experiments revealed the existence of fast-exchanging and strongly solvent-exposed fractions of the helical structures in the two samples Sak and Sak-M26L. These two samples are also thermally less stable with unfolding transition temperatures of 43.7 degrees C (Sak) and 43.5 degrees C (Sak-M26L), respectively. On contrast, Sak-M26A and Sak-G34S/R36G/R43H have a slower hydrogen-deuterium exchange, have a smaller solvent-exposed portion of the helical part, and are more resistant against thermal unfolding; the transition temperatures are 51.7 degrees C and 59.3 degrees C, respectively. The secondary structure analysis was performed by two different approaches, by curve-fitting after band narrowing and by pattern recognition (factor analysis) based upon reference spectra of proteins with known crystal structure. Within the limits of the used methods, we are unable to detect significant differences in the secondary structure of the four variants of Sak. According to the results of the factor analysis, the portions of secondary structure elements were obtained to 16-20% alpha-helix, 28-30% beta-sheet, 23-27% turns, 28-30% irregular (random) and other structure. The sharp differences in the specific plasminogen-activating capacity (Sak, Sak-G34S/R36G/R43H and Sak-M26L are fully active, but Sak-M26A does not form a stable complex with plasminogen) are not reflected in the structural features revealed by the infrared spectra of this study.

Impact of point mutations and amino acid modifications on the structure and stability of peptides and proteins probed by FT-IR spectroscopy

Abstract IR spectroscopy was used to study the impact of amino acid modifications on the association behaviour of β-amyloid peptides (βA4 peptides) and to investigate the effect of point mutations on the secondary structure of the enzyme ribonuclease T1 (RNase T1).

The Thermally Induced Hydrogen Exchange in Different Proteins as Seen by FTIR Spectroscopy

The importance of the flexibility of a protein molecule (“the fourth dimension”) for the proper functioning of the protein can not be over-estimated. One of the most potent methods to study the motility of protein structure is the detection of peptide NH H-exchange [1,2]. The approach is based on the notion that protons are accessible for the solvent catalysts (OH- and H3O+) which cause exchange only if the H-bond the proton is involved in is broken. There are several models describing processes which bring about breakage of intramolecular H-bonds [3]. It has been proposed long ago to use H-D exchange sensitive IR bands to elucidate the flexibility of proteins [4]. Recently we studied extensively H-D exchange processes in several proteins (among them proteins which can not been tuned through the phase transition reversibly) under conditions of a steady and sudden heating of the enzyme solution.

Protein Refolding Triggered by Temperature Jump or Fast Denaturant Dilution: An FTIR Spectroscopic Study

FTIR spectroscopy has been used for the first time to investigate the kinetics of secondary structure formation during the refolding of small globular proteins. The refolding process was induced either by applying a temperature jump on the thermally denatured protein or by rapid dilution of a high concentrated denaturant solution containing the chemically unfolded protein. The experiments were carried out with Ribonuclease A (RNase A) as model system. The dead time of injection and the time resolution of the FTIR spectrometer permitted the observation of refolding kinetics in a time window ranging from milliseconds to several minutes and even hours.