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How does guanidium denature DNA?

How does guanidium denature DNA?


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Guanidium salts like (G-isothiocyanate) disrupt the hydrophobic interactions inside a protein or nucleic acid and denature it. What happens when hydrophobic interactions in DNA are broken? (I don't think it should get ssDNA because guanidium doesn't break hydrogen bonds within molecules)


Guanidium is a chaotrope i.e it increases the entropy in the solution. It doesn't disrupt hydrophobic interactions. Guanidium and urea act by forming hydrogen bonds. They can bond with both the dissolved macromolecule and water. The water molecules are arranged as a shell around the hydrophobic regions so as to contain it, which leads to the reduction of entropy. This containment causes hydrophobic regions to come together and give rise to a pseudo-interaction (hydrophobic interaction. See the figure below). Guanidium interacts with the water shell and disorders it, thereby reducing the hydrophobic effect. Guanidium can also interact with macromolecules directly, by forming hydrogen bonds. In this process they can disrupt the normal H-bonds in the macromolecule.


It is important to realize that the hydrophobic effect is largely due to the exclusion of water from non-polar surfaces, which increases entropy. This effect is a major contributor to the helical structure of DNA, which effectively brings the bases closer together and excludes water from contacting their hydrophobic rings. Chaotropic agents like guanidine thiocyanate increase water entropy by disrupting its hydrogen bonding network (decreasing it's order) and this makes it more favourable for hydrophobic surfaces to be exposed to water, thus denaturation can occur. Implicit in that process is that GITC is a hydrogen bond competitor, which is also evident when looking at its structure. So, in fact, GITC can disrupt the hydrogen bonds between base pairs and denature dsDNA at high enough concentrations.

That all said, we're ignoring a major component of aqueous solutions that can also form hydrogen bonds with the bases: water itself! So we must ask ourselves that if a compound capable of hydrogen bonding (water) is present, what exactly is it that holds helical DNA together? While base pairing certainly does contribute, the major factors are the hydrophobic and base stacking interactions.


Denaturation (biochemistry)

Denaturation is a process in which proteins or nucleic acids lose the quaternary structure, tertiary structure, and secondary structure which is present in their native state, by application of some external stress or compound such as a strong acid or base, a concentrated inorganic salt, an organic solvent (e.g., alcohol or chloroform), radiation or heat. [3] If proteins in a living cell are denatured, this results in disruption of cell activity and possibly cell death. Protein denaturation is also a consequence of cell death. [4] [5] Denatured proteins can exhibit a wide range of characteristics, from conformational change and loss of solubility to aggregation due to the exposure of hydrophobic groups. Denatured proteins lose their 3D structure and therefore cannot function.

Note 1: Modified from the definition given in ref. [1]

Note 2: Denaturation can occur when proteins and nucleic acids are subjected to elevated temperature or to extremes of pH, or to nonphysiological concentrations of salt, organic solvents, urea, or other chemical agents.

Note 3: An enzyme loses its catalytic activity when it is denaturized. [2]

Protein folding is key to whether a globular or membrane protein can do its job correctly it must be folded into the right shape to function. However, hydrogen bonds, which play a big part in folding, are rather weak and thus easily affected by heat, acidity, varying salt concentrations, and other stressors which can denature the protein. This is one reason why homeostasis is physiologically necessary in many life forms.

This concept is unrelated to denatured alcohol, which is alcohol that has been mixed with additives to make it unsuitable for human consumption.


Denaturation

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Denaturation, in biology, process modifying the molecular structure of a protein. Denaturation involves the breaking of many of the weak linkages, or bonds (e.g., hydrogen bonds), within a protein molecule that are responsible for the highly ordered structure of the protein in its natural (native) state. Denatured proteins have a looser, more random structure most are insoluble. Denaturation can be brought about in various ways—e.g., by heating, by treatment with alkali, acid, urea, or detergents, and by vigorous shaking.

The original structure of some proteins can be regenerated upon removal of the denaturing agent and restoration of conditions favouring the native state. Proteins subject to this process, called renaturation, include serum albumin from blood, hemoglobin (the oxygen-carrying pigment of red blood cells), and the enzyme ribonuclease. The denaturation of many proteins, such as egg white, is irreversible. A common consequence of denaturation is loss of biological activity (e.g., loss of the catalytic ability of an enzyme).

The Editors of Encyclopaedia Britannica This article was most recently revised and updated by Adam Augustyn, Managing Editor, Reference Content.


Three-Dimensional Structure of Proteins

4.6 Denaturation

Denaturation of a native protein may be described as a change in its physical, chemical, or biological properties. Mild denaturation may disrupt tertiary or quaternary structures, whereas harsher conditions may fragment the chain. Mild denaturation normally is a reversible process. Some of the changes in properties that may be caused by denaturation are as follows:

decreased solubility (often but not invariably)

alteration in the internal structure and arrangement of peptide chains that does not involve breaking the peptide bonds (e.g., separation of subunits of oligomeric proteins)

disrupted secondary structure (e.g., loss of helical structure)

increased chemical reactivity of functional groups of amino acids, particularly ionizable and sulfhydryl groups (e.g., shift of pK values)

increased susceptibility to hydrolysis by proteolytic enzymes

decrease or total loss of the original biological activity and

loss of crystallizability.

Studies by Anfinsen of the reversible denaturation of the pancreatic enzyme ribonuclease prompted the hypothesis that secondary and tertiary structures are derived inclusively from the primary structure of a protein ( Figures 4-11 and 4-12 ). RNase A, which consists of a single polypeptide chain of 124 amino acid residues, has four disulfide bonds. Treatment of the enzyme with 8 M urea, which disrupts noncovalent bonds, and β-mercaptoethanol, which reduces disulfide linkages to cysteinyl residues, yields a random coil conformation.

FIGURE 4-11 . Amino acid sequence of bovine ribonuclease A. The molecule contains four disulfide bridges.

FIGURE 4-12 . Denaturation and renaturation of ribonuclease A.

However, if both reagents are removed and the cysteinyl residues are allowed to oxidize and re-form disulfide bonds, of the 105 different possible intramolecular combinations of disulfide linkages, only the four correct bonds form, and the denatured enzyme returns to its original, biologically active structure ( Figure 4-12 ). These experiments are taken as proof that the primary structure (which is genetically controlled) determines the unique three-dimensional structure of a protein. However, as described below, it is now known that the folding of some proteins is assisted by other proteins.


3 basic steps of PCR process

The polymerase chain reaction is a three step cycling process consisting of defined sets of times and temperatures.

3 basic PCR steps include:

Each of these polymerase chain reaction steps is repeated 30–40 times (cycles).

In the course of each cycle, the PCR reaction mixture is transferred between three temperatures.

Profile of 3 basic PCR steps

During successive cycles of basic PCR steps (denaturation, annealing, and extension) all the new strands will act as DNA templates causing an exponential increase in the amount of DNA produced.

Each cycle doubles the number of DNA molecules (amplicons) amplified from the DNA template.

First polymerase chain reaction step – DNA denaturation

The first of 3 PCR steps is a denaturation step.

During the denaturation step, the hydrogen bonds that hold together the two strands of the double-stranded nucleic acids are broken and the strands unwind from each other.

This process releases single-stranded DNA to act as templates in the final PCR extension step.

The denaturation temperature is above 90°C (usually 94°C) and the time is up to one minute (usually 30 seconds).

Polymerase chain reaction steps

Second polymerase chain reaction step – DNA Primer annealing

At the annealing step, DNA primers line up on exposed nucleotide sequences at the DNA target according to base-pairing rules. This is a typical temperature-dependent DNA : DNA hybridization reaction and has to be optimized.

This is the only temperature in a PCR cycle steps that can be widely varied.

The temperature depends on the exact sequence and length of the primers.

Usually, the PCR reaction mixture is cooled down to 40–60°C. However, annealing temperatures for DNA templates with a high GC content can be as high as 72°C (the normal temperature of the extension step).

The temperature for this PCR step is chosen for the optimum binding of the DNA primers to the correct DNA template and depends on primer’s melting temperature. The wrong annealing temperature can result in false products, or in no detectable products at all.

Since the primers are relatively short, and at high molar concentrations, duration of the annealing step is around 30 seconds.

At this step, the annealed oligonucleotides provide a free 3’ hydroxyl group for Taq polymerase and act as primers for synthesis of nucleic acids.

Final polymerase chain reaction step – DNA synthesis

The last of 3 basic PCR steps is called extension or elongation step.

It is the DNA synthesis step and carried out by a thermostable DNA polymerase (usually Taq polymerase).

The temperature of the elongation step is usually set at 72°C. It is slightly below the optimum for Taq polymerase.

The Taq polymerase produces complementary DNA strands starting from the primers.

The synthesis proceeds at approximately 1000 bases per minute.

Therefore, to amplify a DNA template that is 500 bases in length, under normal conditions a time of the PCR extension step should be at least 30 seconds.

Usually, PCR extension time is 30 seconds for every 500 bp (base pair) of product.


Thermodynamics of denaturant-induced unfolding of a protein that exhibits variable two-state denaturation

Free energy changes (DeltaG(degrees)(N-->D)) obtained by denaturant-induced unfolding using the linear extrapolation method (LEM) are presumed to reflect the stability differences between native (N) and denatured (D) species in the absence of denaturant. It has been shown that with urea and guanidine hydrochloride (GdnHCl) some proteins exhibit denaturant-independent (DeltaG(degrees)(N-->D)). But with several other proteins urea and GdnHCl give different (DeltaG(degrees)(N-->D)) values for the same protein, meaning that the free energy difference between N and D is not the only contribution to one or both (DeltaG(degrees)(N-->D)) values. Using beta1, a mutant form of the protein G B1 domain, we show that both urea- and GdnHCl-induced denaturations are two-state and reversible but that the denaturants give different values for (DeltaG(degrees)(N-->D)). While spectral observables are sensitive to the shift between N and D states (between states effect), they are not sensitive to denaturant-induced changes that occur within the individual N and D states (within state effect). By contrast, nonspectral observables such as Stokes radius and thermodynamic observables such as proton uptake/release are often sensitive to both "between states" and "within state" effects. These observables, along with spectral measurements, provide descriptions of urea- and GdnHCl-induced denaturation of beta1. Our results suggest that in the predenaturation concentration range GdnHCl changes the free energy of the native ensemble in a nonlinear manner but that urea does not. As with RNase A and beta-lactoglobulin, beta1 exhibits variable two-state behavior with GdnHCl-induced denaturation in that the free energy of the native ensemble in the predenaturation zone changes (varies) with GdnHCl concentration in a nonlinear manner.


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2E: Laboratory Determination of ΔG° of Protein Unfolding

  • Contributed by Henry Jakubowski
  • Professor (Chemistry) at College of St. Benedict/St. John's University
  • from a graph of an observable vs [denaturant], determine the DG0 in the absence of denaturant for the N to D transition
  • from a graph of an observable vs T, determine the DG0, DH0, and DS0 at a given temperature for the N to D transition

HOW TO CALCULATE D G O FOR (N ightleftharpoons D) FROM PROTEIN DENATURATION CURVES:

Multiple methods can be used to investigate the denaturation of a protein. These include UV, fluorescence, CD, and viscosity measurement. In all these methods the dependent variable (y) is measured as a function of the independent variable, which is often temperature (for thermal denaturation curves) or denaturant (such as urea, guanidine hydrochloride) concentration. From these curves we would like to calculate the standard free energy of unfolding ( D G O ) for the protein (for the reaction (N ightleftharpoons D) ). The denaturation curves usually show a sigmoidal, cooperative transition from the native to the denatured state. The dependent variable can also be normalized to show fractional denaturation (fD). An idealized example of an experimental denaturation curve is shown in the figure below:

Figure: DENATURATION CURVE FOR PROTEINS

A more realistic denaturation curve might show a linear change in the values of the dependent variable (fluorescence intensity for example) for values of temperature or denaturant concentration well below that at which the protein starts to unfold, or above that at which it is unfolded. In these cases, the mathematical analysis, presented below, is a bit more complicated.

For each curve, the value of y (either A280, Fluorescence intensity, viscosity, etc) can be thought of as the sum contributed by the native state and from the denatured state, which are present in different fractional concentrations from 0 - 1. Hence the following equation should be reasonably intuitive.

where fN is the fraction native and yN is the contribution to the dependent variable y from the native state, and fD is the fraction denatured and yD is the contribution to the dependent variable y from the denatured state. Conservation give equation 2.

[1 = f_N + f_D:: extrm:: f_N = 1 - f_D ag<2>]

Substituting 2 into 1 gives:

Rearranging this equation gives

Notice the right hand side of the equations contains variables that are easily measured.

By substituting 2 and 3 into the expression for the equilibrium constant for the reaction (N ightleftharpoons D) we get:

[ Delta G^circ = -RT ln, K_ = -RT, ln left( dfrac <1 - f_D> ight ) ag<5a>]

Remember that D G O (and hence Keq) depends only on the intrinsic stability of the native vs denatured state for a given set of conditions. They vary as a function of temperature and solvent conditions. At low temperature and low urea/guanidine HCl concentration, the native state is favored, and for the (N ightleftharpoons D) transition, D G O > 0 (i.e. denaturation is NOT favored). At high temperature and urea/guanidine HCl concentration, the denatured state is favored, and D G O < 0. At some value of temperature or urea/guanidine concentration, both the native and denatured state would be equally favored. At this point, Keq = 1 and D G O = 0. If temperature is the denaturing agent, the temperature at this point is called the melting point (Tm) of the protein, which is analogous to the Tm (in the heat capacity vs temperature graphs) for the gel to liquid crystalline phase transition of phospholipid vesicles.

Figure: gel to liquid crystalline phase transition of phospholipid vesicles

Figure: gel to liquid crystalline phase transition of phospholipid vesicles

Ordinarily, at a temperature much below the Tm for the protein or at a low urea concentration, so little of the protein would be in the D state that it would be extremely difficult to determine the concentration of the protein in the D state. Hence it would be difficult to determine the Keq or D G o for the reaction (N ightleftharpoons D) . However, in the range where the protein denatures (either with urea or increasing temperature), it is possible to measure fD/fN.and hence D G o at each urea or temperature.

Denaturation with chemical perturbants (such as urea): Calculation of D G o for (N ightleftharpoons D) in the absence of urea

A plot of D G o vs [urea] is linear, and given by the following equation, which should be evident from the beginning figure in this section:

5b. D G 0D = D G 0D(w/o urea) - m[urea]

Although it is nice to know the Keq and D G 0 for the (N ightleftharpoons D) transition in the presence of various urea concentrations, it would be even more useful to determine those parameters in the absence of urea, that is, under "physiological conditions". A comparison of the calculated values of D G 0 in the absence of urea for a series of similar proteins (such as those varying by a single amino acid prepared by site-specific mutagenesis of the normal or wild-type gene, would indicate how the mutants were stabilized or destabilized compared to the wild-type protein. In experimental cases in which the denaturant is a substance such as urea or guanidine HCl, the D G 0D for the protein in the absence of denaturant (i.e in water) can be determined by extrapolating the straight line to [urea] = 0. Admittedly, this is a long extrapolation, but with high quality data and a high correlation coefficient for the linear regression analysis of the best-fit line, reasonable values can be obtained.

Denaturation with heat: Calculation of D H o and D S o for (N ightleftharpoons D) at room temperature

Keq values can be calculated from thermal denaturation curves in the same way as described above using urea as a denaturant by monitoring change in an observable (spectra signal for example) vs temperature. Knowing Keq (denoted KD below since at the moment my program won't allow me to change it) and DH0, D S 0 can be calculated from equation 8 below as a semi-log plot of lnKeq vs 1/T is a straight line with a slope of - D H0R and a y intercept of + D S0 /R.

[ Delta G^o = Delta H^o - T Delta S^o = -RT, ln , K_D ag<6>]

Hence from Equations (6) and (8) is should be evident that all the major thermodynamics constants (D G0 , D H0 and D S0 ) for the (N ightleftharpoons D) transition can be calculated from thermal denaturation curves. Equation (9) shows that the derivative of equation (8) with respect to 1/T (i.e. the slope of equation 8 plotted as lnKD vs 1/T) is indeed - D H0 /R. Equation (8) is the van 't Hoff equation, and the calculated value of the enthalpy change is termed the van 't Hoff enthalpy, D H0vHoff . Equation (10) calculates the derivative of lnKeq with respect to T instead of 1/T. This method assumes that the denaturation is cooperative (no intermediates) and that DH0 is independent of temperature over the narrow range of temperature in which the protein cooperatively unfolds.

This equation will be useful later when we compare the enthalpies calculated using the van 't Hoff equation with those determined directly using differential scanning calorimetry by analyzing a plot of Cp vs T. (Note that the area under the Cp vs T curve as the protein transitions to the unfolded state has units of kcal. The DH0 for the unfolding is inversely proportional to the width of the curve.) .

In contrast to the long extrapolation of the D G0 vs [urea] to [urea] = 0 to get D G0 (the y intercept) in the absence of urea, which has some physical meaning, extrapolation of the straight line from the van 't Hoff plot from equation 8 to get D S0 /R, the y intercept, has little meaning since the 1/T value at the y intercept is 0, which occurs when T approaches infinity. D S0 can be calculated at any reasonable temperature from the the calculated value of D G0 at that temperature and the calculated D H0vHoff .

Thumbnail: Structure of human hemoglobin. The proteins &alpha and &beta subunits are in red and blue, and the iron-containing heme groups in green. From PDB: 1GZX​. (GNU Proteopedia Hemoglobin).


Abstract

Free energy changes ( ) obtained by denaturant-induced unfolding using the linear extrapolation method (LEM) are presumed to reflect the stability differences between native (N) and denatured (D) species in the absence of denaturant. It has been shown that with urea and guanidine hydrochloride (GdnHCl) some proteins exhibit denaturant-independent . But with several other proteins urea and GdnHCl give different values for the same protein, meaning that the free energy difference between N and D is not the only contribution to one or both values. Using β1, a mutant form of the protein G B1 domain, we show that both urea- and GdnHCl-induced denaturations are two-state and reversible but that the denaturants give different values for . While spectral observables are sensitive to the shift between N and D states (between states effect), they are not sensitive to denaturant-induced changes that occur within the individual N and D states (within state effect). By contrast, nonspectral observables such as Stokes radius and thermodynamic observables such as proton uptake/release are often sensitive to both “between states” and “within state” effects. These observables, along with spectral measurements, provide descriptions of urea- and GdnHCl-induced denaturation of β1. Our results suggest that in the predenaturation concentration range GdnHCl changes the free energy of the native ensemble in a nonlinear manner but that urea does not. As with RNase A and β-lactoglobulin, β1 exhibits variable two-state behavior with GdnHCl-induced denaturation in that the free energy of the native ensemble in the predenaturation zone changes (varies) with GdnHCl concentration in a nonlinear manner.


Denaturation (biochemistry)

Denaturation is the alteration of a protein shape through some form of external stress (for example, by applying heat, acid or alkali), in such a way that it will no longer be able to carry out its cellular function.

Denatured proteins can exhibit a wide range of characteristics, from loss of solubility to communal aggregation.

Proteins are very long strands of amino acids linked together in specific sequences.

A protein is created by ribosomes that "read" codons in the gene and assemble the requisite amino acid combination from the genetic instruction, in a process known as translation.

The newly created protein strand then undergoes post-translational modification in which additional atoms or molecules are added, for example copper, zinc, iron.

Once this post-translational modification process has been completed, the protein begins to fold (spontaneously, and sometimes with enzymatic assistance), curling up on itself so that hydrophobic elements of the protein are buried deep inside the structure and hydrophilic elements end up on the outside.

The final shape of a protein determines how it interacts with its environment.


Watch the video: Denaturation and Renaturation of DNA (October 2022).