Interaction of rat testis protein, TP, with nucleic acids in vitro. Fluorescence quenching, UV absorption, and thermal denaturation studies

The nucleic acid binding properties of the testis protein, TP, were studied with the help of physical techniques, namely, fluorescence quenching, UV difference absorption spectroscopy, and thermal melting. Results of quenching of tyrosine fluorescence of TP upon its binding to double-stranded and denatured rat liver nucleosome core DNA and poly(rA) suggest that the tyrosine residues of TP interact/intercalate with the bases of these nucleic acids. From the fluorescence quenching data, obtained at 50 mM NaCl concentration, the apparent association constants for binding of TP to native and denatured DNA and poly(rA) were calculated to be 4.4 X 10(3) M-1, 2.86 X 10(4) M-1, and 8.5 X 10(4) M-1, respectively. UV difference absorption spectra upon TP binding to poly(rA) and rat liver core DNA showed a TP-induced hyperchromicity at 260 nm which is suggestive of local melting of poly(rA) and DNA. The results from thermal melting studies of binding of TP to calf thymus DNA at 1 mM NaCl as well as 50 mM NaCl showed that although at 1 mM NaCl TP brings about a slight stabilization of the DNA against thermal melting, a destabilization of the DNA was observed at 50 mM NaCl. From these results it is concluded that TP, having a higher affinity for single-stranded nucleic acids, destabilizes double- stranded DNA, thus behaving like a DNA-melting protein.

Irreversible Thermal Denaturation Of Bovine Pancreatic Ribonuclease-A

The isolation and characterization of the products formed during the irreversible thermal denaturation of enzyme RNAase-A are described. RNAase-A, when maintained in aqueous solution at pH 7.0 and 70° for 2 h, gives soluble products which have been fractionated by gel filtration on Sephadex G-75 into four components. These components are designated RNAase-At1, RNAase-At2, RNAase-At3 and RNAase-At4 according to the order of their elution from Sephadex G-75. RNAase-At4 shows the same specific activity towards yeast RNA as native RNAase-A and is virtually indistinguishable from it by the physical methods employed. However, chromatography on CM-cellulose separates it into three components that show the same u.v. spectra and specific activity towards yeast RNA as native RNAase-A. RNAase-At1, RNAase-At2and RNAase-At3 are all structurally altered derivatives of RNAase-A and they exhibit low specific activity (5–10%) towards yeast RNA. In the presence of added S-protein, all these derivatives show greatly enhanced enzymic activity. RNAase-At1 and RNAase-At2 are polymers, covalently crosslinked by intermolecular disulfide bridges; whereas RNAase-At3 is a monomer. Physical studies such as 1H-n.m.r., sedimentation analysis, u.v. absorption spectra and CD spectra reveal that RNAase-At3 is a unfolded derivative of RNAase-A. However, it is seen to possess sufficient residual structure which gives rise to a low but easily detectable enzymic activity.

A simple model for protein thermal denaturation

Whether proteins denature in all-or-none fashion or in a continuous fashion is as yet an unresolved problem. The all-or-none process implies that while the process of denaturation is going on, only two kinds of protein molecules can exist. One is completely unchanged and the other is altered. The altered protein molecules are indistinguishable. Underlying the 'continuum' models is the assumption that all the chains in a protein globule undergo similar changes so that it is enough to consider a single chain.

Study of thermal fluctuations during thermal denaturation of DNA

In this paper we address the fundamental issue of temperature fluctuation during the thermal denaturation (or the unzipping of the two strands on heating) of double stranded (ds) DNA. From our experiments we observe the presence of extremely high thermal fluctuations during DNA denaturation. This thermal fluctuation is several orders higher than the thermal fluctuation at temperatures away from the denaturation temperature range. This fluctuation is absent in single stranded (ss) DNA. The magnitude of fluctuation is much higher in heteropolymeric DNA and is almost absent in short homopolymeric DNA fragments. The temperature range over which the denaturation occurs (i.e., over which the thermal fluctuation is large) depends on the length of the DNA and is largest for the longest DNA.

A simple model for protein thermal denaturation

Whether proteins denature in all-or-none fashion or in a continuous fashion is as yet an unresolved problem. The all-or-none process implies that while the process of denaturation is going on, only two kinds of protein molecules can exist. One is completely unchanged and the other is altered. The altered protein molecules are indistinguishable. Underlying the 'continuum' models is the assumption that all the chains in a protein globule undergo similar changes so that it is enough to consider a single chain.

Thermal Fluctuation Spectroscopy of DNA Thermal Denaturation

We have developed the technique of thermal fluctuation spectroscopy to measure the thermal fluctuations in a system. This technique is particularly useful to study the denaturation dynamics of biomolecules like DNA. Here we present a study of the thermal fluctuations during the thermal denaturation (or melting) of double-stranded DNA. We find that the thermal denaturation of heteropolymeric DNA is accompanied by large, non-Gaussian thermal fluctuations. The thermal fluctuations show a two-peak structure as a function of temperature. Calculations of enthalpy exchanged show that the first peak comes from the denaturation of AT rich regions and the second peak from denaturation of GC rich regions. The large fluctuations are almost absent in homopolymeric DNA. We suggest that bubble formation and cooperative opening and closing dynamics of basepairs causes the additional fluctuation at the first peak and a large cooperative transition from a partially molten DNA to a completely denatured state causes the additional fluctuation at the second peak.

The denaturation of β-lactoglobulin-A at pH 2

The denaturation of β-lactoglobulin-A by heat and guanidine hydrochloride at pH 2 has been investigated. The effect of ethylene glycol on the thermal denaturation at this pH has also been studied. The conditions of the experiments have been chosen so as to eliminate complications arising out of disulfide interchange, changes in the degree of association of the protein during denaturation, and intermolecular aggregation. The physical parameters characterizing the denatured states of the protein which are produced by heat and guanidine hydrochloride have been determined. The thermodynamic parameters for these transitions have been estimated using a two-state hypothesis in each case. Both the physical and thermodynamic parameters indicate that the heat-denatured state of β-lactoglobulin-A retains about 15-20% of residual structure which is destroyed on adding guanidine hydrochloride.

Interactions of L-serine at the active site of serine hydroxymethyltransferases: induction of thermal stability

Serine hydroxymethyltransferase (SHMT), EC 2.1.2.1, exhibits broad substrate and reaction specificity. In addition to cleaving many 3-hydroxyamino acids to glycine and an aldehyde, the enzyme also catalyzed the decarboxylation, transamination and racemization of several substrate analogues of amino acids. To elucidate the mechanism of interaction of substrates, especially L-serine with the enzyme, a comparative study of interaction of L-serine with the enzyme from sheep liver and Escherichia coli, was carried out. The heat stability of both the enzymes was enhanced in the presence of serine, although to different extents. Thermal denaturation monitored by spectral changes indicated an alteration in the apparent T, of sheep liver and E. coli SHMTs from 55 +/- 1 degrees C to 72 +/- 3 degrees C at 40 mM serine and from 67 +/- 1 degrees C to 72 +/- 1 degrees C at 20 mM serine, respectively. Using stopped flow spectrophotometry k values of (49 +/- 5)(.)10(-3) s(-1) and (69 +/- 7).10(-3) s(-1) for sheep liver and E. coli enzymes were determined at 50 mM serine. The binding of serine monitored by intrinsic fluorescence and sedimentation velocity measurements indicated that there was no generalized change in the structure of both proteins. However, visible CD measurements indicated a change in the asymmetric environment of pyridoxal 5'-phosphate at the active site upon binding of serine to both the enzymes. The formation of an external aldimine was accompanied by a change in the secondary structure of the enzymes monitored by far UV-CD spectra. Titration microcalorimetric studies in the presence of serine (8 mM) also demonstrated a single class of binding and the conformational changes accompanying the binding of serine to the enzyme resulted in a more compact structure leading to increased thermal stability of the enzyme.

Thermodynamic characterization of the conformational stability of the homodimeric protein, pea lectin

The conformational stability of the homodimeric pea lectin was determined by both isothermal urea-induced and thermal denaturation in the absence and presence of urea. The denaturation profiles were analyzed to obtain the thermodynamic parameters associated with the unfolding of the protein. The data not only conform to the simple A(2) double left right arrow 2U model of unfolding but also are well described by the linear extrapolation model for the nature of denaturant-protein interactions. In addition, both the conformational stability (Delta G(s)) and the Delta C-p for the protein unfolding is quite high, at about 18.79 kcal/ mol and 5.32 kcal/(mol K), respectively, which may be a reflection of the relatively larger size of the dimeric molecule (M-r 49 000) and, perhaps, a consequent larger buried hydrophobic core in the folded protein. The simple two-state (A(2) double left right arrow 2U) nature of the unfolding process, with the absence of any monomeric intermediate, suggests that the quaternary interactions alone may contribute significantly to the conformational stability of the oligomer-a point that may be general to many oligomeric proteins.

Photoinduced DNA and Protein Cleavage Activity of Ferrocene-Conjugated Ternary Copper(II) Complexes

Ferrocene-conjugated ternary copper(II) complexes [Cu(L)(B)](ClO4)(2), where L is FcCH(2)N(CH2Py)(2) (Fc = (eta(5)-C5H4)Fe-II(eta(5)-C5H5)) and B is a phenanthroline base, viz., 2,2'-bipyridine (bpy, 1), 1, 10-phenanthroline (phen, 2), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 3), and dipyrido[3,2-a:2',3'-c]phenazine (dppz, 4), have been synthesized and characterized by various spectroscopic and analytical techniques. The bpy complex 1, as its hexafluorophosphate salt, has been structurally characterized by X-ray crystallography. The molecular structure shows the copper(II) center having an essentially square-pyramidal coordination geometry in which L with a pendant ferrocenyl (Fc) moiety and bpy show respective tridentate and bidentate modes of binding to the metal center. The complexes are redox active, showing a reversible cyclic voltammetric response of the Fc(+)-Fc couple near 0.5 V vs SCE and a quasi-reversible Cu(II)-Cu(I) couple near 0.0 V. Complexes 2-4 show binding affinity to calf thymus (CT) DNA, giving binding constant (K-b) values in the range of 4.2 x 10(4) to 2.5 x 10(5) M-1. Thermal denaturation and viscometric titration data suggest groove binding and/or a partial intercalative mode of binding of the complexes to CT DNA. The complexes show good binding propensity to the bovine serum albumin (BSA) protein, giving K-BSA values of similar to 10(4) M-1 for the bpy and phen complexes and similar to 10(5) M-1 for the dpq and dppz complexes. Complexes 2-4 exhibit efficient chemical nuclease activity in the presence of 3-mercapto-propionic acid (MPA) as a reducing agent or hydrogen peroxide (H2O2) as an oxidizing agent. Mechanistic studies reveal formation of hydroxyl radicals as the reactive species. The dpq and dppz complexes are active in cleaving supercoiled (SC) pUC19 DNA on photoexposure to visible light of different wavelengths including red light using an argon-krypton mixed gas ion laser. Mechanistic investigations using various inhibitors reveal the fort-nation of hydroxyl radicals in the DNA photocleavage reactions. The dppz complex 4, which shows efficient photoioduced BSA cleavage activity, is a potent multifunctional model nuclease and protease in the chemistry of photodynamic therapy (PDT) of cancer.

Importance of amino-terminus in maintenance of oligomeric structure of cytosolic serine hydroxymethyltransferase

The role of the amino and carboxyl-terminal regions of cytosolic serine hydroxymethyltransferase (SHMT) in subunit assembly and catalysis was studied using six amino-terminal (lacking the first 6, 14, 30, 49, 58, and 75 residues) and two carboxyl-terminal (lacking the last 49 and 185 residues) deletion mutants. These mutants were constructed from a full length cDNA clone using restriction enzyme/PCR-based methods and overexpressed in Escherichia coli. The overexpressed proteins, des-(A1-K6)-SHMT and des-(A1- W14)-SHMT were present in the soluble fraction and they were purified to homogeneity. The deletion clones, for des-(A1–V30)-SHMT and des-(A1–L49)-SHMT were expressed at very low levels, whereas des-(A1–R58)-SHMT, des-(A1–G75)-SHMT, des-(Q435–F483)-SHMT and des-(L299-F483)-SHMT mutant proteins were not soluble and formed inclusion bodies. Des-(A1–K6)-SHMT and des-(A1–W14)-SHMT catalyzed both the tetrahydrofolate-dependent and tetrahydrofolate-independent reactions, generating characteristic spectral intermediates with glycine and tetrahydrofolate. The two mutants had similar kinetic parameters to that of the recombinant SHMT (rSHMT). However, at 55 °C, the des-(A1–W14)-SHMT lost almost all the activity within 5 min, while at the same temperature rSHMT and des-(A1–K6)-SHMT retained 85% and 70% activity, respectively. Thermal denaturation studies showed that des-(A1–W14)-SHMT had a lower apparent melting temperature (52°C) compared to rSHMT (56°C) and des-(A1–K6)-SHMT (55 °C), suggesting that N-terminal deletion had resulted in a decrease in the thermal stability of the enzyme. Further, urea induced inactivation of the enzymes revealed that 50% inactivation occurred at a lower urea concentration (1.2 ± 0.1 M) in the case of des-(A1–W14)-SHMT compared to rSHMT (1.8 ±0.1 M) and des-(A1–K6)-SHMT (1.7 ±0.1 M). The apoenzyme of des-(A1- W14)-SHMT was present predominantly in the dimer form, whereas the apoenzymes of rSHMT and des-(A1–K6)-SHMT were a mixture of tetramers (≈75% and ≈65%, respectively) and dimers. While, rSHMT and des-(A1–K6)-SHMT apoenzymes could be reconstituted upon the addition of pyridoxal-5'-phosphate to 96% and 94% enzyme activity, respectively, des-(A1–W14)-SHMT apoenzyme could be reconstituted only upto 22%. The percentage activity regained correlated with the appearance of visible CD at 425 nm and with the amount of enzyme present in the tetrameric form upon reconstitution as monitored by gel filtration. These results demonstrate that, in addition to the cofactor, the N-terminal arm plays an important role in stabilizing the tetrameric structure of SHMT.

Importance of the amino terminus in maintenance of oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

The Role Of The Amino And Carboxyl-Terminal Regions Of Cytosolic Serine Hydroxymethyltransferase (SHMT) In Subunit Assembly And Catalysis Was Studied Using Sis Amino-Terminal (Lacking The First 6, 14, 30, 49, 58, And 75 Residues) And Two Carboxyl-Terminal (Lacking The Last 49 And 185 Residues) Deletion Mutants. These Mutants Were Constructed From A Full Length Cdna Clone Using Restriction Enzyme/PCR-Based Methods And Overexpressed In Escherichia Coli. The Overexpressed Proteins, Des-(A1-K6) SHMT And Des-(A1-W14)-SHMT Were Present In The Soluble Fraction And They Were Purified To Homogeneity. The Deletion Clones, For Des-(A1-V30)-SHMT And Des-(A1-L49)-SHMT Were Expressed At Very Low Levels, Whereas Des-(A1-R58)-SHMT, Des-/A1-G75)-SHMT, Des-(Q435-F483)-SHMT And Des-(L299-F483)-SHMT Mutant Proteins Were Not Soluble And Formed Inclusion Bodies. Des-(A1-K6)-SHMT And Des-(A1-W14)-SHMT Catalyzed Both The Tetrahydrofolate-Dependent And Tetrahydrofolate-Independent Reactions, Generating Characteristic Spectral Intermediates With Glycine And Tetrahydrofolate. The Two Mutants Had Similar Kinetic Parameters To That Of The Recombinant SHMT (Rshmt). However, At 55 Degrees C, The Des-(A1-W14)-SHMT Lost Almost All The Activity Within 5 Min, While At The Same Temperature Rshmt And Des-(A1-K6)-SHMT Retained 85% And 70% Activity, Respectively. Thermal Denaturation Studies Showed That Des-(A1-W14)-SHMT Had A Lower Apparent Melting Temperature (52 Degrees C) Compared To Rshmt (56 Degrees C) And Des-(A1-K6)-SHMT (55 Degrees C), Suggesting That N-Terminal Deletion Had Resulted In A Decrease In The Thermal Stability Of The Enzyme. Further Urea Induced Inactivation Of The Enzymes Revealed That 50% Inactivation Occurred At A Lower Urea Concentration (1.2+/-0.1 M) In The Case Of Des-(A1-W14)-SHMT Compared To Rshmt (1.8+/-0.1 M) And Des-(A1 -K6)-SHMT (1.7+/-0.1 M). The Apoenzyme Of Des-/A1-K6)-SHMT Was Present Predominantly In The Dimer Form, Whereas The Apoenzymes Of Rshmt And Des-(A1-K6)-SHMT Were A Mixture Of Tetramers (Approximate To 75% And Approximate To 65%, Respectively) And Dimers. While, Rshmt And Des-(A1-K6)-SHMT Apoenzymes Could Be Reconstituted Upon The Addition Of Pyridoxal-5'-Phosphate To 96% And 94% Enzyme Activity, Respectively Des-(A1-W14)-SHMT Apoenzyme Could Be Reconstituted Only Upto 22%. The Percentage Activity Refined Correlated With The Appearance Of Visible CD At 425 Nm And With The Amount Of Enzyme Present In The Tetrameric Form Upon Reconstitution As Monitored By Gel Filtration. These Results Demonstrate That, In Addition To The Cofactor, The N-Terminal Arm Plays An Important Role In Stabilizing The Tetrameric Structure Of SHMT.