Victor N. Orlov

Temperature-induced selective death of the C-domain within angiotensin-converting enzyme molecule

Somatic angiotensin‐converting enzyme (ACE) consists of two homologous domains, each domain bearing a catalytic site. Differential scanning calorimetry of the enzyme revealed two distinct thermal transitions with melting points at 55.3 and 70.5°C. which corresponded to denaturation of C‐ and N‐domains, respectively. Different heat stability of the domains underlies the methods of acquiring either single active N‐domain or active N‐domain with inactive C‐domain within parent somatic ACE. Selective denaturation of C‐domain supports the hypothesis of independent folding of the two domains within the ACE molecule. Modeling of ACE secondary structure revealed the difference in predicted structures of the two domains, which, in turn, allowed suggestion of the region 29–133 in amino acid sequence of the N‐part of the molecule as responsible for thermostability of the N‐domain.

Expression and Functional Characterization of the First Bacteriophage-Encoded Chaperonin

ABSTRACT Chaperonins promote protein folding in vivo and are ubiquitously found in bacteria, archaea, and eukaryotes. The first viral chaperonin GroEL ortholog, gene product 146 (gp146), whose gene was earlier identified in the genome of bacteriophage EL, has been shown to be synthesized during phage propagation in Pseudomonas aeruginosa cells. The recombinant gp146 has been expressed in Escherichia coli and characterized by different physicochemical methods for the first time. Using serum against the recombinant protein, gp146s native substrate, the phage endolysin gp188, has been immunoprecipitated from the lysate of EL-infected bacteria and identified by mass spectrometry. In vitro experiments have shown that gp146 has a protective effect against endolysin thermal inactivation and aggregation, providing evidence of its chaperonin function. The phage chaperonin has been found to have the architecture and some properties similar to those of GroEL but not to require cochaperonin for its functional activity.

Thermal stability and aggregation of creatine kinase from rabbit skeletal muscle. Effect of 2-hydroxypropyl-beta-cyclodextrin.

Effect of 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) on thermal aggregation of creatine kinase from rabbit skeletal muscle (RMCK) at 48 degrees C has been studied using dynamic light scattering. An increase in the duration of the lag period on the kinetic curves of aggregation, registered as an increment of the light scattering intensity in time, has been observed in the presence of HP-beta-CD. It has been shown that the initial parts of the dependences of the hydrodynamic radius (R(h)) of the protein aggregates on time follow the exponential law. The reciprocal value of parameter t(2R) (t(2R) is the time interval over which the R(h) value is doubled) was used to characterize the rate of aggregation. A 10-fold decrease in the 1/t(2R) value was observed in the presence of 76mM HP-beta-CD. Judging from the data on the kinetics of RMCK inactivation and the data on differential scanning calorimetry of RMCK, HP-beta-CD does not affect the rate of RMCK unfolding.

Differential scanning calorimetric study of the complexes of modified myosin subfragment 1 with ADP and vanadate or beryllium fluoride

SummaryThe effects of various modifications of rabbit skeletal myosin subfragment 1 on thermal denaturation of subfragment 1 in ternary complexes with Mg-ADP and orthovanadate (Vi) or beryllium fluoride (BeFx) have been studied by differential scanning calorimetry. It has been shown that specific modifications of SH1 group of Cys-707 by different sulfhydryl reagents, trinitrophenylation of Lys-83, and reductive methylation of lysine residues promote the decomposition of the S1·ADP·Vi complex and change the character of structural transitions of the subfragment 1 molecule induced by the formation of this complex, but they have much less or no influence on subfragment 1 thermal stability in the S1·ADP·BeFx complex. Thus, the differential scanning calorimetric studies on modified subfragment 1 preparations reveal a significant difference between S1·ADP·Vi and S1·ADP·BeFx complexes. It is suggested that S1·ADP·Vi and S1·ADP·BeFx complexes represent structural analogues of different transition states of the ATPase cycle, namely the intermediate states S1**·ADP·Pi and S1*·ATP, respectively. It is also proposed that during formation of the S1·ADP·Vi complex the region containing both Cys-707 and Lys-83 plays an important role in the spread of conformational changes from the active site of subfragment 1 ATPase throughout the structure of the entire subfragment 1 molecule. In such a case, the effects of reductive methylation of lysine residues on the subfragment 1 structure in the S1·ADP·Vi complex are related to the modification of Lys-83.

Interaction of myosin subfragment 1 with F‐actin studied by differential scanning calorimetry

The thermal unfolding of the myosin subfragment 1 (S1) and of filamentous actin (F‐actin) in their strong complex obtained in the presence of ADP was studied by differential scanning calorimetry (DSC). It is shown that in the acto‐S1 complexes S1 and F‐actin melt separately, and thermal transitions of each protein can be easily followed. Interaction of S1 with F‐actin significantly increases S1 thermal stability and also affects the thermal stability of F‐actin. Although S1 unfolds at much lower temperature than F‐actin, the molecules of S1 remain bound to F‐actin even after full denaturation. Under these conditions S1 may induce cross‐linking between actin filaments. It is concluded that DSC studies on the acto‐S1 complexes offer a new and promising approach to investigate the structural changes which occur in the myosin head and in F‐actin due to their interaction.

Macroscopic Aggregation of Tobacco Mosaic Virus Coat Protein

The relationship between processes of thermal denaturation and heat-induced aggregation of tobacco mosaic virus (TMV) coat protein (CP) was studied. Judging from differential scanning calorimetry “melting” curves, TMV CP in the form of a trimer–pentamer mixture (“4S-protein”) has very low thermal stability, with a transition temperature at about 40°C. Thermally denatured TMV CP displayed high propensity for large (macroscopic) aggregate formation. TMV CP macroscopic aggregation was strongly dependent on the protein concentration and solution ionic strength. By varying phosphate buffer molarity, it was possible to merge or to separate the denaturation and aggregation processes. Using far-UV CD spectroscopy, it was found that on thermal denaturation TMV CP subunits are converted into an intermediate that retains about half of its initial α-helix content and possesses high heat stability. We suppose that this stable thermal denaturation intermediate is directly responsible for the formation of TMV CP macroscopic aggregates.

Thermal unfolding of phosphorylating d-glyceraldehyde-3-phosphate dehydrogenase studied by differential scanning calorimetry

Thermal unfolding parameters were determined for a two-domain tetrameric enzyme, phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and for its isolated NAD(+)-binding domain. At pH 8.0, the transition temperatures (t(max)) for the apoforms of the native Bacillus stearothermophilus GAPDH and the isolated domain were 78.3 degrees C and 61.9 degrees C, with calorimetric enthalpies (DeltaH(cal)) of 4415 and 437 kJ/mol (or 30.7 and 22.1 J/g), respectively. In the presence of nearly saturating NAD(+) concentrations, the t(max) and the DeltaH(cal) increased by 13.6 degrees C and by 2365 kJ/mol, respectively, for the native apoenzyme, and by 2.8 degrees C and 109 kJ/mol for the isolated domain. These results indicate that interdomain interactions are essential for NAD(+) to produce its stabilizing effect on the structure of the native enzyme. The thermal stability of the isolated NAD(+)-binding domain increased considerably upon transition from pH 6.0 to 8.0. By contrast, native GAPDH exhibited greater stability at pH 6.0; similar pH-dependencies of thermal stability were displayed by GAPDHs isolated from rabbit muscle and Escherichia coli. The binding of NAD(+) to rabbit muscle apoenzyme increased t(max) and DeltaH(cal) and diminished the widths of the DSC curves; the effect was found to grow progressively with increasing coenzyme concentrations. Alkylation of the essential Cys149 with iodoacetamide destabilized the apoenzyme and altered the effect of NAD(+). Replacement of Cys149 by Ser or by Ala in the B. stearothermophilus GAPDH produced some stabilization, the effect of added NAD(+) being basically similar to that observed with the wild-type enzyme. These data indicate that neither the ion pairing between Cys149 and His176 nor the charge transfer interaction between Cys149 and NAD(+) make any significant contribution to the stabilization of the enzymes native tertiary structure and the accomplishment of NAD(+)-induced conformational changes. The H176N mutant exhibited dramatically lower heat stability, as reflected in the values of both DeltaH(cal) and t(max). Interestingly, NAD(+) binding resulted in much wider heat capacity curves, suggesting diminished cooperativity of the unfolding transition.

A comparative differential scanning calorimetric study of tobacco mosaic virus and of its coat protein ts mutant

The differential scanning calorimetry (DSC) ‘melting curves’ for virions and coat proteins (CP) of wild‐type tobacco mosaic virus (strain U1) and for its CP ts mutant ts21–66 were measured. Strain U1 and ts21–66 mutant (two amino acid substitutions in CP: I21 → T and D66 → G) differ in the type of symptoms they induce on some host plants. It was observed that CP subunits of both U1 and ts21–66 at pH 8.0, in the form of small (3–4S) aggregates, possess much lower thermal stability than in the virions. Assembly into the virus particles resulted in a DSC melting temperature increase from 41 to 72°C for U1 and from 38 to 72°C for ts21–66 CP. In the RNA‐free helical virus‐like protein assemblies U1 and ts21–66 CP subunits had a thermal stability intermediate between those in 3–4S aggregates and in the virions. ts21–66 helical protein displayed a somewhat lower thermal stability than U1.

Thermal inactivation, denaturation and aggregation of mitochondrial aspartate aminotransferase.

A comparative study of thermal denaturation and inactivation of aspartate aminotransferase from pig heart mitochondria (mAAT) has been carried out (10 mM Na phosphate buffer, pH 7.5). Analysis of the data on differential scanning calorimetry shows that thermal denaturation of mAAT follows the kinetics of irreversible reaction of the first order. The kinetics of thermal inactivation of mAAT follows the exponential law. It has been shown that the inactivation rate constant (k(in)) is higher than the denaturation rate constant (k(den)). The k(in)/k(den) ratio decreases from 28.8+/-0.1 to 1.30+/-0.09 as the temperature increases from 57.5 to 77 degrees C. The kinetic model explaining the discrepancy between the inactivation and denaturation rates has been proposed. The size of the protein aggregates formed at heating of mAAT at a constant rate (1 degrees C min(- 1)) has been characterized by dynamic light scattering.

Changes in the thermal unfolding of p‐phenylenedimaleimide‐modified myosin subfragment 1 induced by its ‘weak’ binding to F‐actin

Differential scanning calorimetry (DSC) was used to analyze the thermal unfolding of myosin subfragment 1 (S1) with the SH1 (Cys‐707) and SH2 (Cys‐697) groups cross‐linked by N,N′‐p‐phenylenedimaleimide (pPDM‐S1). It has been shown that F‐actin affects the thermal unfolding of pPDM‐S1 only at very low ionic strength, when some part of pPDM‐S1 binds weakly to F‐actin, but not at higher ionic strength (200 mM KCl). The weak binding of pPDM‐S1 to F‐actin shifted the thermal transition of pPDM‐S1 by about 5°C to a higher temperature. This actin‐induced increase in thermal stability of pPDM‐S1 was similar to that observed with ‘strong’ binding of unmodified S1 to F‐actin. Our results show that actin‐induced structural changes revealed by DSC in the myosin head occur not only upon strong binding but also on weak binding of the head to F‐actin, thus suggesting that these changes may occur before the power‐stroke and play an important role in the motor function of the head.