Basics of Proteins

8/12/2019 Basics of Proteins

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1.PROTEIN STRUCTURE

Note: this document represent a more detailed discussion of information given in the syllabus.

I. CHARACTERISTICS OF PROTEINS

In conjunction with their diversity in function, there are many structures of proteins.Nevertheless, several general features are shared.

A. Composition and Size

Proteins are complex macromolecules of high molecular weight, ranging from 5,000 toseveral million Daltons. They are composed of carbon, hydrogen, nitrogen, oxygen, and usuallysulfur; some contain small amounts of other elements. Proteins may be simple or may beconjugated with other non-protein substances (for example, with lipids to form lipoproteins).

B. The Amino Acids

Proteins are polymers; the monomeric units are amino acids. Most proteins are formed fromthe same set of 20amino acids. A few proteins contain non-standard amino acids that are derivedfrom the standard set. Proteins contain from about 50 to several thousand amino acid residues.

The characteristics and structures of amino acids are important to understanding proteinfunction and many disease processes.

The carbons are labeled using Greek letters starting at thefirst or carbon. Attached to the carbon is an amino group andan acid group; hence the name -amino acid. The carbons inthe side-chain, R, are labeled serially with Greek letters (, ,

etc.). Sometimes amino acids are written in the un-ionized form(for example, the NH3+is shown as NH2, and COO-is shownas COOH).

All the amino acids except glycine are chiral and thereforeproteins are chiral. The chiral amino acids contain one or more chiral carbons, in which fourdifferent substituents are attached to the chiral center. Amino acids are normally present in theL configuration; some amino acids of the D configuration are found in the cell walls of somebacteria and other sources. Note that the D or L refers to configuration in relation to referencecompounds such as glyceraldehyde. By another convention, chiral compounds are called R(Latin: rectus, right) or S(Latin: sinister, left)

compounds. Again, R and Srefer to the spatialconfiguration.

A chiral compound isoptically active in that asolution containing thecompound can rotate theplane of polarized light.

NH3+

C

R

-OOC

H

COO-

C

R

+H3N

H

!"#!$#

!%#!%#

!"# !$#

R amino Acid (or D) S amino acid (or L)

Fig. 2 The chiral character of amino acids.

H3N+ C C

R

O-

OH

Fig. 1 The -amino acid. Rrefers to the side chain thatdistinguishes the amino acids.


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Rules for assigning the value to substituents for a chiral molecule are as follows: Of the fouratoms attached to an asymmetric carbon, the atom of higher atomic number is assigned a highervalue. If two atoms are the same, comparison of the next atoms attached to each is made. Adouble (or triple) bond counts as two (or three) of the atomic number for the attached atom. Todetermine the configuration of the amino acid alanine (R=CH3), for example, values areassigned to the atoms that are attached to the asymmetric carbon: NH2-1, COOH-2, R-3, andH-4. Envision the molecule as a steering wheel with H-4 in back. Go from 1 to 2 to 3. If thesteering wheel turns to the right, it is an R amino acid; if it turns to the left, it is an S amino acid.

The key to deciding the polar character of the amino acids is to examine their atomicstructure. The parts of amino acids with hydrocarbons(CH, CH2, CH3) are hydrophobic; theparts with oxygensand nitrogensare hydrophilic.

There are different ways to classify the amino acids. Here they are classified by theirchemistry. They are sometimes grouped by their partition coefficient between an organic solvent(chloroform) and water, with more hydrophilic amino acids dissolving more readily in water.They can also be classified by their appearance in globular proteins, with more polar amino acidsappearing at the surface.

1. Amino acids with mostly nonpolar side-chains

Alanine Valine

CH3

H

-OOC

+NH3

C

CH

H

-OOC

+NH3

CH3

CH3

C

Leucine Isoleucine Methionine

CH2

H

-OOC

+NH3

CHCH3

CH3

C

CH

H

-OOC

+NH3

CH2

CH3

CH3C

CH2

H

-OOC

+NH3

CH2

S CH3C

Phenylalanine Tryptophan Tyrosine

C

H2

H

-OOC

+NH3

C

C

H

-OOC

+NH3NH

HC

C CH2

C

H2

H

-OOC

+NH3

OHC


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ProlineH

C-OOC

+H 2N

CH2

CH2

CH2

Note that proline has a 5 member ring.

Note that tryptophan and tyrosine have polar portions of their side-chains and that the nonpolarportions of glycine and alanine are small.

2. Polar with neutral side-chains(at pH 7).

Threonine Serine

CH

H

-OOC

+NH3

CH3

OH

C

CH2

H

-OOC

+NH3

OHC

Asparagine Glutamine

C

H

-OOC

+NH3

NH2

O

CH2

C

C

NH2

O

CH2

H

-OOC

+NH3

CH2

C

3. Polar with charged side-chains(at pH 7)

Aspartate (Aspartic acid) Glutamate (Glutamic acid)

C

H

-OOC

+NH3

O-

O

CH2

C

C

O-

O

CH2

H

-OOC

+NH3

CH2

C

Note the relationship of aspartate to asparagine and that of glutamate to glutamine.

Lysine Arginine

CH2

H

-OOC

H3N+

CH2 NH3+CH2 CH2C

CH2

H

-OOC

H3N+

CH2 CCH2 NH

NH2

NH2+

C

Histidine


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C

NH+

CH

HN

CH2

C

H

-OOC

+NH3

4. Cysteine

CH2

H

-OOC

+NH3

SHC

Cysteine behaves as nonpolar when un-charged, and polar when charged (usu-ally above pH 8). Under oxidizing con-ditions (typically outside the cell), cys-teine residues form covalent bonds

through disulfide bridges. The disul-fide can cross link different chains orwithin the same chain, and imparts increased stability to protein structures.

5. Glycine, has a small side-chain that allows it to get into tight spaces.

H

H

-OOC

+NH3

C

Reference: Three-letter abbreviations and one-letter symbols of the amino acids

Amino Acid Codes Amino Acid Codes Amino Acid Codes

3 letter 1 letter 3 letter 1 letter 3 letter 1 letterAlanine Ala A Glycine Gly G Proline Pro PAsparagine Asn N Histidine His H Serine Ser SAspartate Asp D Isoleucine Ile I Threonine Thr TArginine Arg R Leucine Leu L Valine Val VCysteine Cys C Lysine Lys K Tryptophan Trp WGlutamate Glu E Methionine Met M Tyrosine Tyr YGlutamine Gln Q Phenylalanine Phe F

C. The Peptide Bond

The amino acids of a protein are linked by peptideor amide bonds in a head-to-tail fashionto form one or more chains of amino acids. A chainof amino acids is called a polypeptide. The

amino acid sequence, determined by the mRNA sequence is specific for each protein.Note that the formation of the peptide bond between an amino and a carboxyl group has

eliminated their charges. Only the most amino terminal amine and the most carboxyl terminalcarboxyl groups are still present:

S

S

S

S

S S

Fig. 3 Cysteine residues can form disulfide bonds betweenpolypeptides. Each SS indicates the linkage between

the sulfurs of two cysteines.


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+H3N C CO2-

H

R1

+H3N C CO2-

H

R2

C

H

C

H

C

R1

N

H

O

CO2-

R2

+H3N + H2O+

Peptide bond

Fig. 4 Formation of the peptide bond. The carboxyl group of one amino acid combines with the amino group

of another.

The functional properties existing in a protein are contained mainly in the R-groups orside-chains. The three-dimensional structure or conformationof proteins is also determined bythe properties of the amino acid residues. Each protein has a unique amino acid sequence and adistinct three-dimensional structure. Some chains of amino acids will form a protein domain thatis globular. These proteins, which include enzymes and transport proteins, are usually water-soluble. Non-polar amino acids predominate on the inside of globular water-soluble proteins;polar ones predominate on the outside. Other chains of amino acids can form aggregates and arerelatively insoluble in many aqueous solutions. These fibrousproteins are often elongated andinclude proteins such as collagen, which is abundant in skin.

The polarity of the side-chain of an amino acid can dictate its role within a protein. A water-soluble protein has mostly amino acids with hydrophilic (water-loving) side-chains on thesurface interacting with water. The interior of such proteins is hydrophobic (water-fearing) withthe non-polar side-chains interacting with each other. On the other hand, the part of a protein thatpenetrates a membrane is composed mostly of amino acids with hydrophobic side-chains(membranes are hydrophobic). Likewise, regions of proteins found associated with negativelycharged molecules (such as DNA) have surface amino acids that are positively charged.

Note that the atoms N, O, and S can accept hydrogen bonds, whereas hydrogens attached tothese atoms (NH, OH, SH) can donate hydrogen bonds. Proteinsare rich in hydrogen bonds both the side-chains of polaramino acids and the peptide bond are involved. Hydrogen bondsform when a hydrogen atom is shared between twoelectronegative atoms. The strength of a hydrogen bond isdistance dependent, and maximum strength occurs at shortdistances. The strongest hydrogen bonds contain atoms that arecollinear (in the same line). The local environment may alsoinfluence the strength of a hydrogen bond; nonpolar regions favorhydrogen bond formation.

HX X

- -+ (where X is nitrogen, oxygen, or sulfur)

D. Proteins sometimes contain several domains.The role of proteins in disease is often detected through genetic analysis. For example,

consider the tumor suppressor protein p53, which plays a critical role in protecting cells againstcancer. This protein and its gene have been studied extensively because the loss or malfunctionof p53 contributes to the development of about half of all cancers, including common ones suchas skin, breast, and colon cancer. DNA sequencing has shown frequent missense mutations thatcluster near the center of the gene encoding the protein (Fig. 6).

Fig. 5. Model of insulin. Thehydrogen bonds are shown asdotted lines.

B-chain

A-chain


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5' 3'

transcription, translation

NH2 COOHgene activation Sequence specific DNA binding p53-p53 interaction

gene activation Sequence specific DNA binding p53-p53 interaction

p53 gene

p53 protein

x xxx x x

Figure 6 The p53 gene and protein. Mutational hot spots, which result in changed amino acids, are shown as 'x'on the protein sequence. They cluster in the central DNA binding region of the protein. The protein has regionswith other biochemical functions gene activation and self-association.

p53, a protein of about 400 amino acid residues, can be isolated and analyzedbiochemically.The three functional domains are organized into three distinct and independentfolding units of 50 to 200 amino acids each (Fig. 7). Each domain behaves much like a separateprotein, and a separate biochemical function is associated with each domain. One domain bindsDNA in a sequence specific manner, another activates certain genes, and another associates withitself. The DNA-binding domain contacts specific base sequences on DNA. The transcriptional

activation domain signals assembly the basal transcription factors for the transcription of geneslocated near the p53 DNA-binding sites. The oligomerization domain enhances the strength andspecificity of DNA binding because four binding domains are available instead of just one.

Small proteins, containing less than 200 amino acids, usually have only one domain and onefunction. Protein domains are the fundamental functional and three-dimensional structural unitsof a protein. The amino acid chains of each domain are folded into specific three-dimensionalshapes. They are connected by non-structured stretches of amino acids.

Typical of multi-domain proteins, each domain is encoded by groups of one or more exons.Various segments or functional domains of p53 protein have been studied individually. Thecentral domain is responsible for forming a complex with a specific sequence of DNA. The N-terminal region is responsible for transactivation, and coordinates with basal transcription factorswhereas the C-terminus is responsible for p53-p53 interactions. The spatial arrangement of theatoms in the domains, the three-dimensional structure, has been determined by X-raycrystallographic methods or by nuclear magnetic resonance (NMR) methods.

E. The amino acids of a protein form a specific structure with a specific function.

Figure 7 The folded units (domains) of the p53 protein.

Oligomerization

DNA-binding (4x)Transcriptional activation (4x)


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A protein is very specific in its function. The function and specificity of a protein aredependent on the amino acid sequence and the three-dimensional structure. Consider, forexample, DNA recognition. Different DNA sequences present different patterns of hydrogenbond acceptors (oxygens, nitrogens), hydrogen bond donors (NH groups), and hydrophobiccontacts (CH3groups). For example, the part of the GC base pair that is accessible through the

major groove of DNA shows two hydrogen bond acceptors from the guanine (Figure 8). In anAT base pair, the pattern is a hydrogen bond donor from the adenine, a hydrogen bond acceptorfrom the thymine, and a methyl group from the thymine. An essential feature of most DNA-binding proteins, such as p53, is a segment of protruding protein that fits into the major grooveof DNA. Analogous to the hydrogen bonding that occurs between base-pairs of DNA, aminoacid side-chain residues of the protein segment can form hydrogen bonds to the edge of thebase-pairs. The amino acids of a specific DNA-binding protein are positioned to interact moststrongly with one a particular DNA sequence. Usually between ten and twenty hydrogen bondsform between a DNA-binding protein and DNA. The correct positioning for the DNA-readingsegment is stabilized by electrostatic interactions (positive charges attract negative ones)between a neighboring segment of the protein and the sugar-phosphate backbone of the DNA.Figure 8 shows how the specificity of function derives from the specific three-dimensional

structure of the protein in the case of p53.

Motion, or dynamics is an important aspect of protein structure. For example, p53 may bindto slightly different DNA sites with similar affinity, but will adopt slight different structures. Inthe absence of the target, proteins typically are more mobile and show a range of structures thatinclude the target-bound conformation.


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III. CLASSIFICATION OF PROTEIN STRUCTURE

The structure of proteins can be considered at four levels.

A. Primary structure. The sequence of amino acids in a polypeptide (or chain, or protein)

B. Secondary structure. The steric relationships involving hydrogen bonds and adjacentamino acids in a polypeptide. These conformations of the polypeptide may be coiled orextended. Their structure is frequently repeating (-helix, pleated sheet, collagen helix).

C. Tertiary structure. The steric relationship of non-adjacent amino acids in a polypeptide.These conformations may involve the folding of globular proteins into compact structuresutilizing interactions of charged amino acid groups as well as van der Waals interactions.

D. Quaternary structure. The structure formed by the interaction and relationship of separateidentical or non-identical polypeptides.

A. Primary Structure

Fig. 8 Contacts to DNA made by p53. Left: A segment of the protein reaches into the major groove of the DNA.This segment is stabilized in part by binding to a zinc ion (shown as a sphere). A part of the protein-DNA interface iscircled and expanded on the right: here one can clearly see the hydrogen bonds made by arginine 280 to one of the

base-pairs. Note that hydrogen bonds by the neighboring residue, aspartate 281, position the side-chain in the correctorientation for reading the DNA base-pair, and that aspartate 281 is in turn held in place by an arginine residue(residue 273). On the lower right of the figure, the positive charge of the arginine side-chain can be seen to make afavorable electrostatic interaction with the phosphate of the DNA backbone. For clarity, only some of the side-chains

are shown.


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The primary structure of proteins refers to the sequence of amino acid residues. Byconvention, they are named from the N-terminal to the C-terminal end. For example, thistetrapeptide is named alanyl-glycyl-histidyl-leucine (Ala-Gly-His-Leu):

+H3N CH

CH3

C

O

N C

H2

C

O

N CH C

O

N

CH2

CH COO-

H2C

CH

H3C

H H H

NH

HN

+CH3

Fig. 9 Structure of a tetrapeptide, alanyl-glycyl-histidyl-leucine.

Simple peptides (dipeptides, tripeptides, tetrapeptides, etc.) are short amino acid chainslinked by peptide bonds. Peptides can be produced by partial hydrolysis of the peptide chains of

proteins. Alternatively, some are synthesized as such in the body.

Except for peptide bonds, thepeptides have essentially freerotation about their bonds.Peptide bonds undergo resonanceand are planar:

The C=O and the C-N bonds have both single and double bond characteristics. The partial double bond

character in the C-N bond prevents rotation about this bond and restricts the amide group to a planar and almostalways transconfiguration. Thus, only other bonds permit rotation in a polypeptide.

Although peptides usually have little defined global conformation, some, such as peptidehormones, are physiologically active.Larger protein fold to maximize weak, non-covalent forces such as hydrogen bonds,hydrophobic interaction, electrostatic bonds, and van der Waals forces.

Hydrogen bonds. Hydrogen bonds form when a hydrogen atom is shared between twoelectronegative atoms. The local environment influences the strength of a hydrogen bond; thosein nonpolar regions are stronger than those at the surface of a protein.

Hydrophobic interactions. Each amino acid residue in a polypeptide, especially the nonpolarones, induces the formation of a solvation shell by water, and thus imparts some order to thewater. When two nonpolar groups of an unstructured polypeptide come together, the surfacearea exposed to water is reduced, and therefore the ordering of the water is reduced. It is calledthe hydrophobiceffect. In thermodynamic terms, this is an increase in the entropy of the water,which lends to a favorable, stable system. The interaction of (mostly) nonpolar residues leads toa small loss of entropy; however, the entropy gained from disordering of the water solvationshell is relatively large. Thus, the gain in protein stability from hydrophobic forces is not from

-

+

H

O

CNR

R

H

O

CNR

R

Fig. 10 Resonance of the peptide bond keeps the atoms coplanar.


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the intrinsic attraction of hydrophobic residues, but mostly from a loss of orderingof the water(solvent) shell. The net result is the sequestering of nonpolar residues of the protein from theaqueous environment.

Electrostatic bonds are interactions between atoms of opposite charges. The strongestelectrostatic bonds occur in regions of low polarity, such as the interior of the protein. They arealso known as ionic bonds or salt bridges.

Van der Waals forces. Two atoms are optimally separated by the sum of their van der Waalsradii. Atoms have fluctuating electrical charges and therefore local regions of partial positiveand partial negative charge. A weak bonding interaction exists when two atoms approach due tothe attraction between the transient partial charges. This interaction is strongly dependent ondistance at closest distances there is repulsion between electron clouds. At long distances, thevan der Waals force becomes negligible.

Note that these different forces are related to each other. For example, the hydrogen bond isreally the sum of a van der Waals force and the electrostatic force between the electronegative

atom (usually N or O) and the partially positive hydrogen. The hydrophobic effect is the mostpoorly understood force, but it is clear the solvation shell for a protein involves hydrogen bondforces between water molecules and between water and the protein.

B. Secondary Structure

The secondary structure refers to a local structural element that stretches of amino acids canfold into. There are usually 10 to 40 amino acids in a secondary element. The chemistry of theamino acids, such as planarity of the peptide bond, suggests that only a few conformations canbe adopted by a polypeptide. The distinct conformations are governed by a number of differentatomic forces, including electrostatic interactions and hydrogen bonds. The different secondary

elements are characterized by differences in hydrogen bonding pattern.

Proposing that a polypeptide will fold to maximize the number of hydrogen bonds, LinusPaulingpredicted that polypeptides will contain the -helixand the -sheet (also known as thepleatedsheet). Three-dimensional structural determination of protein structures hasexperimentally verified these predictions.

C

Trans-amide in peptide

H

H

C

O

N

O

C

N

HR

C

H

C

O

N

R

O

C

N

H

C

H

RH

Planar amide group

Peptide bond Rotate here

cis-amide in uracil

N

N

O

H

H

O

Fig. 11 The configuration of the peptide bond and conformational mobility in peptides.


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Fig. 12 Model of the -helix. Left: Note that theoxygen of the carbonyl group is hydrogen-bonded(shown as a dashed line) to the HN of the aminoacid four residues down the chain. Below: Theamino acid side-chains stick out radially from thecore.

PROGLY

ARG+

ASP-

ARG+

ARG+

THR

GLU-

GLU-

GLU

1. The -helix

In -helical structures the amino acid residues are arranged in a spiral with ~3.5 amino acidsper turn. The helix rises 1.5 per residue and has 5.4 between each turn. Note that the atomsin hydrogen bonds are collinear and the hydrogen bonds are strong.

The -helix is right handed with respect to the direction of the spiral. Note that the side-chains of amino acids adjacent in sequence are spatially proximate. Therefore, if two or more

amino acid adjacent in sequence have ionized side-chains of like charge, for example, the -helixis not energetically favorable. This sequence of amino acids would adopt another secondarystructure. The five-member ring of prolinealso cannot adopt the bond conformations needed tofit into the middle of a helix it is known as ahelixbreaker.

In close-up views of protein structure where allindividual bonds between the atoms are shown, thepath of the polypeptide is indicated by a tracethrough the backbone atoms (the nitrogen, the carbon and the carbonyl carbon). The helix, forexample, is shown as a ribbon in the shape of a

cylinder (Fig. 12). In overall views of proteinstructure, the helix can also be shown by acylinder itself.

2. The sheet

In the conformation or pleated sheet, thepolypeptides are much more extended than in the -helix. Hydrogen bonding is betweenstretches of amino acid residues rather than within a continuous stretch of amino acid residues.

C

N

CH-R

C

N

R-HC

C

N

N

C

R-HC

N

C

CH-R

N

C

O

H

O

H

O

H

H

O

H

O

H

O

Fig. 13 Model of a (antiparallel) -sheet.


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The chains may be parallel (i.e., with chains running from N-terminal to C-terminal in onedirection) or antiparallel (with chains running in opposite directions).

Again, in close-up views of protein structure as in Fig. 13, the bonds between the atoms areshown. In overviews of the protein, the individual strands of the sheet are shown by broadarrows, with the arrowhead on the C-terminal amino acid of the strand (pointing away from theN-terminal amino acid).

A typical protein will contains these elements of secondary structure connected by a series ofbends (known as hairpin bends or -turns), non-regular loops, and disorderedpeptide regions.

Some groups of secondary elements, called super-secondary elementsor protein structural motifs, occur inmany proteins. For example, the core of a protein may contain two sheets that constitute a sandwich.

C. Tertiary Structure

The tertiary structure is the description ofthe conformational fold of a protein, or how

the secondary elements of a protein interactwith one another. They can be described bestby showing a picture (Fig. 14). Many water-soluble proteins approximate a sphere.

Not only is protein chiral at the aminoacid level, but globally adoptsstereospecificity in its three-dimensionalshape. Many drugs that bind to proteins arealso chiral compounds. The synthesis of thesedrugs often results in a mixture of the R and Sforms (a racemic mixture), with one formbeing active, and the other form either

inactive or dangerous.Fig. 14 The DNA-binding domain of p53 bound toDNA. The major part of the domain comprises two sheets that stack face to face across an extendedhydrophobic core. The Zn2+ion is below the left endof the helix at the top of the protein. The zinc isliganded by cysteine 176, histidine 179, cysteine 238,and cysteine 242 and thus holds together parts of the

protein chain that are distant (about 60 residues) insequence. Note the complementary fit between the

protrusion of the protein and the major groove of theDNA.

D. Quaternary Structure

The quaternary structure is the interaction and relationship of separate (identical ornon-identical) polypeptides. Only proteins that have more than one polypeptide chain havequaternary structure. p53, for example, exists as a tetramer in nature, i.e., has four polypeptides.The four DNA-binding domains are connected to the oligomerization domain by flexible, non-structured loops. Although Figures 8 and 14 show only one subunit binding to a pentamer DNAsequence, full-length p53 binds as a tetramer to four closely spaced DNA sequences of 5 base-


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pairs each (i.e., 20 base-pairs are contacted in total).

IV. THE EFFECT OF MUTATIONS ON PROTEIN STRUCTURE

The changing of one amino acid to another often has little consequence for the function of aprotein when the substitution conserves the nature of the amino acid. When a polar amino acid is

substituted by a non-polar amino acid, however, or vice versa, or the charge of the amino acidchanges, there can be a dramatic effect on function.

Mutations in the p53 DNA-binding domain have been studied in detail. One class ofmutations involves residues that contact the DNA. Failure to bind DNA by these mutants can beattributable to a loss of a critical DNA interaction. These mutants have little change in three-dimensional (secondary, tertiary) structure. Another class of mutations involves residues that areimportant for the correct fold of the DNA-binding domain. Loss of DNA binding by thesemutants can be attributed to structural defects. Certain mutant p53 proteins have been shown tobe stabilized with small molecules (i.e., potential new drugs).

Basics of Proteins

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