Biochem CH 8-12

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Polysaccharides are..

• long chains of monosaccharides linked by glycosidic bonds, serving as energy storage or structural components in organisms.

• sometimes, the repeating units of polysaccharides are modified. The polymers can be either linear or branched. The physical and chemical properties of polysaccharides (rigidness, solubility in water, etc.) depend on their structure

Homopolysaccharides

contain only one type of monosaccharides

Heteropolysaccharide

more than one monosaccharide type constitutes the polymer,

Structural Polysaccharides

provide rigidness for cells and tissues

Storage Polysaccharides

serve as secondary energy sources

Signaling Polysaccharides

attached to membrane proteins and lipids and participate in intercellular recognition events

 

____ and ____ are the most widely spread structural polysaccharides on Earth.

Cellulose, chitin

Heteropolysaccharide vs. Homopolysaccharide

____ is the structural component of plant cell walls. It accounts for about __ of all plant dry matter. It is also produced by algae and bacteria

Cellulose, 33%

Cellulose represents a linear chain of glucose residues connected via ___________

β-1,4-glycosidic bonds

Cellulose is a homodimer of ______

D-glucose

Why is cellulose insoluble in water?

Despite the hydrophilicity of cellulose fibers, this polymer is insoluble in water due to the rigidity and tightness of the fibrils’ structure. This occurs because the intermolecular hydrogen bonds between the cellulose chains create a strong structural matrix, preventing water from penetrating the fiber.

_____is the process of breaking down cellulose into smaller polysaccharides cellodextrins and into glucose.

Cellulolysis

• Because cellulose molecules bind strongly to each other, cellulolysis is relatively difficult compared to the breakdown of other polysaccharides.

____ is a long-chain linear homopolymer consisting of N-acetylglucosamine units connected via β-1,4-glycosidic bonds It is a chemical analog of cellulose with the only structural difference in the replacement of OH group at C₂ by N-acetylamino group.

Chitin

___is the structural component of cell wall of fungi and of exoskeletons of crustaceans, insects and spiders.

Chitin

• Due to its flexibility, strength, and ability to be degraded in the human body, chitin is used as surgical thread in medicine.

• In addition, chitin is known to accelerate wound healing.

• In agriculture, it is used as a fertilizer, which stimulates the immune system of plants.

   

Starch is a principal energy source produced by plants. It is composed of glucopyranose monomers linked by ____ bonds. It is a mixture of two glucose polymers: ________ and ________

α-glycosidic, α-amylose (20–25%), amylopectin (75–80%)

______ is a linear polymer of up to a thousand glucose molecules joined by α-1,4-glycosidic bonds

α-Amylose

• structural isomer of cellulose with the only difference in the configuration at the anomeric centers

• due to this configuration, the polymeric chain of α-amylose assumes a helical rather than a linear structure

• presence of this helical structure in starch accounts for its blue staining in reaction with iodine solution, a well-known test for starch

Amylopectin is a branched glucose polymer. Its linear fragments are ___glucose polymers, while in the branching points glucose units are connected via _____ bonds. The branching usually occurs every 24–30 units. The resulting tree-like structure has only one hemiacetal anomeric carbon, which is called the ______ ___

a(1→4), α-1,6-glycosidic, reducing end.

To digest starch obtained from the diet, humans and other animals have  special enzymes called _____ which hydrolyzes α(1→4) glycosidic bonds.

amylases

______ which enables long-term energy storage in animals, is a more branched structural analog of amylopectin. The branch points occur every 8–12 glucose units.

Glycogen

________ also known as mucopolysaccharides, are unbranched heteropolysaccharides that consist of disaccharide repeating unit. The repeating unit is composed of a residue of uronic or iduronic acids, while the other is a glucosamine or galactosamine residue

Glycosaminoglycans

_______ ____ (hyaluronate or hyaluronan) is a major structural component of connective, epithelial, and neural tissues. It consists of D-glucuronic acid and D-N-acetylglucosamine linked via alternating β(1→4) and β(1→3) glycosidic bonds.

Hyaluronic acid

________ ______ differs from hyaluronic acid only in that N-acetylglucosamine residue is replaced with N-acetylgalactosamine residue, and its either C4 or C6 hydroxyl group is sulfated

Chondroitin sulfate

• Long chains (over 100 monomeric residues) of chondroitin sulfate with OH groups sulfated at variable positions are usually found attached to proteins as part of proteoglycans.

______ _____contains residues of iduronic acid, which is an epimer of glucuronic acid found in chondroitin sulfate, and N-acetylgalactosamine-4-sulfate residues

This glycosaminoglycan takes part in blood coagulation and wound repair.

Dermatan sulfate

_____ a variably and heavily sulfated glycosaminoglycan. It contains an average charge of 2.5 sulfate groups per disaccharide unit, which is composed of sulfated iduronate and glucosamine residues. It has the highest negative charge density of any known biological molecule

Heparin

• naturally-occurring anticoagulant, which acts by preventing the formation of clots and extension of existing clots in blood. Its properties make it be used in medicine for anticoagulation.

the structure determination of a polysaccharide following aspects have to be elucidated:

1. nature and molar ratios of the monosaccharide building blocks;

2. positions of the glycosidic linkages;

3. distinction of furanosidic and pyranosidic forms;

4. anomeric configuration;

5. monomer sequences and identification of repeating units;

6. position and nature of OH–modifications such as O–phosphorylation or O–sulfation.

Heat de-denatured DNA exhibits _____ UV absorbance, which is called the _____

increased; hyperchromic effect

• 40% increase

The DNA helix is stabilized by

hydrogen bonds between complementary bases, stacking interactions, and favorable electrostatic interactions (presence of counterions)

The sugar pucker is ___ in the ___ form

C2’-endo for pyrimindes and C3’-endo for purines: Z-DNA

Which of the following ‘assays’ would be most specific for a particular protein?

Radioimmunoassay

Which of the following amino acids would be last to elute at pH 8.0 from an anion-exchange column?

glutamic acid

The pK₁, pK₂, and pK_R of the amino acid histdine are 1.8, 9.3, and 6.0, respectively. The pK₁, pK₂, and pK_R of the amino acid arginine are 1.8, 9.0, and 12.5, respectively. You have a mixture of histidine and arginine, how would you try to separate these two amino acids?

cation exchange chromatography at pH 9

Which of the following substances cannot be used to cleave peptide bonds in polypeptides?

2-mercaptoethanol

The positive charge on proteins in electrospray ionization mass spectrometry is the result of

protonated side chains of Arg, His and Lys residues

You have purified a new peptide hormone. To determine its amino acid sequence you have digested the polypeptide with trypsin and in a separate reaction you have cleaved the polypeptide with cyanogen bromide.

 

Cleavage with trypsin yielded 5 peptides that were sequenced by Edman degradation as shown in the following.

 

1. Ser─Leu

2. Asp─Val─Arg

3. Val─Met─Glu─Lys

4. Ser─Gln─Met─His─Lys

5. Ile─Phe─Met─Leu─Cys─Arg

 

Cleavage with cyanogen bromide yielded 4 peptides that were sequenced by Edman degradation:

 

1. His─Lys─Ser─Leu

2. Asp─Val─Arg─Val─Met

3. Glu─Lys─Ile─Phe─Met

4. Leu─Cys─Arg─Ser─Gln─Met

 Determine the identity of the N-terminal amino acid after reconstructing the intact protein.

Asp

In vivo DNA is synthesized in the process of ___ ______

DNA Replication

• performed by DNA polymerase which use a single-stranded DNA as a template for the synthesis of the complementary strand

• DNA polymerases, elongate a primer, which synthesized by primase

• 2 new new DNA molecules (replicas) are obtained as a result of the replication

semi-conservative replication mode

the newly synthesized double-stranded DNAs contain one strand from the parent molecule and one new strand

DNA synthesis begins at the ____→ AT-rich to facilitate melting of the double helix

Separation of the two DNA strands at the origin is accomplished by __ ______

_________ relieve topological stress in the helical DNA structure created upon separation of the DNA strands

Unwound strands are kept single-stranded through their binding by special _______________

The ends of the single-stranded loop formed at the origin are called replication forks

Synthesis of both daughter strands is _________

DNA synthesis begins at the origins → AT-rich to facilitate melting of the double helix

Separation of the two DNA strands at the origin is accomplished by DNA helicases

Topoisomerases relieve topological stress in the helical DNA structure created upon separation of the DNA strands

Unwound strands are kept single-stranded through their binding by special single-stranded DNA binding proteins

The ends of the single-stranded loop formed at the origin are called replication forks

Synthesis of both daughter strands is semicontinuous

leading strand

the strand that is synthesized continuously

lagging strand

strand that is synthesized discontinuously

the synthesis of of the daughter strands is semicontinuous what does that mean?

It means that one strand (the leading strand) is synthesized continuously, while the other strand (the lagging strand) is synthesized in short fragments (Okazaki fragments) that are later joined together.

Why does a “nick” occur in DNA replication?

A “nick” occurs in DNA replication due to the process of joining Okazaki fragments on the lagging strand, where the enzyme DNA ligase seals the gaps between fragments that remain unconnected

_______ allows DNA replication to be very accurate

proof-reading

• removal of mispaired nucleotides after incorporation

• removal of mispaired nucleotides after replication

Cellular RNA are synthesized by _____

transcription

the info about RNA nucleotide sequence is stored in a ___

gene

Only 1 strand of __ is transcribed during a single transcription process

DNA

DNA dependent RNA polymerase

enzyme that govern RNA synthesis in vivo; the transcription of RNA from a DNA template, synthesizing RNA strands complementary to the DNA sequence

Prokaryotes have __ type of RNA polymerase and Eukaryotes have _ types of RNA polymerases

1; 3

Why is transcription less accurate than replication?

Transcription is less accurate than replication because RNA polymerase has a lower fidelity than DNA polymerase, resulting in a higher rate of errors when synthesizing RNA from a DNA template - there is also no proofreading

What makes up the RNA secondary structure?

The RNA secondary structure is formed by base pairing between nucleotides, creating structures such as hairpins, loops, and bulges, which help stabilize the RNA molecule.

What makes up the RNA tertiary structure?

The RNA tertiary structure is composed of the overall three-dimensional arrangement of nucleotides that results from interactions between the RNA's secondary structure elements, including hydrogen bonding, stacking interactions, and metal ion coordination, leading to its functional conformation.

mRNA (messenger RNA)

transfer the genetic information stored in DNA to the ribosomes, the “factories” for protein synthesis

rRNA (Ribosomal RNA)

are the components of ribosomes

tRNA (transfer RNA)

Transfer RNAs (tRNA) deliver amino acids to a growing polypeptide chain at the ribosomal site during protein synthesis

miRNA (Micro RNA)

post-transcriptional regulators that bind to complementary sequences of mRNA and inhibit translation

siRNA (small interfering RNA)

participate in regulation of gene expression,

spliceomes

post-translational modification of mRNA

Bacterial ribosomes contain _ rRNAs

3

What makes up the 50s subunit of rRNA

5s rRNA (120 nucleotides), 23s rRNA (2900 nucleotides)

What makes up the 30s subunit

16s rRNA (1540 nucleotides)

Double stranded RNA form a ___

helix

• 11 BP per turn

• 3.1 nm pitch

• 16.7º

2º structure of tRNA

76-90 nucleotides long, cloverleaf

Force stabilizing nucleic acid structures

Hydrogen bonds, base stacking interactions, and ionic interactions

Why are G-C rich DNA more thermally stable?

cations shield the negative charges of nucleic acids

Nucleic acids that are rich in guanine are capable of forming 4-stranded structures called ____

G-quadruplexes

the ability of RNA molecules to store transmit, and duplicate genetic information, as well as biocatalysts and regulators of gene expression (riboswitches) provides some basis for ______

RNA world theory

• early stages of evolution and pre-cellular life depended on RNA

B-DNA

• right handed

• 10 base pairs per turn

• 3.4 nm pitch (rise per turn)

• major groove: 2.2 nm

• minor groove: 1.2 nm

A DNA

• right handed

• 11.6 base pairs per turn

• 3.4 nm pitch (rise per turn)

Z DNA

• left-handed

• 12 base pairs per turn

• 4.4 nm pitch (rise per turn)

• C3’ endo/syn → purines

• C2’ endo/anti→ pyrimindines

• formed by alternating purine/pyrimidine sequences: (Gc)n, (AC)n, (GT)n

Linking number formula

L = T + W

twist (T)

the number of helical turns in a DNA double strand

writhing number (W)

the number of superhelical turns

linking number (L)

the number of time one DNA strand winds around a complementary strand

T is __ for right-handed turns

positive

W<0 for ____ DNA + right-handed superhelical turns

negative super coiled DNA

L is constant for ____ circles

closed

• defined only for a DNA with fixed ends, is an integer #

supercoiled DNA is more ____ so it has greater mobility in gel

compact

DNA transformations that alleviate topological constraints in (-) supercoiled DNA

• melting of DNA fragments

• cruciform structure formation

• formation of short stretches of Z-DNA

  • B-DNA→Z-DNA unwinds (-) superhelical turns

Find L in relaxed DNA w/ 2080 base pairs

L = 200

Biological functions of proteins

include catalysis, structural support, transport, regulation, and antibodies

what does the binding site in a protein do?

allow protein to bind to other proteins, nucleic acids, or small molecules

• it’s formed when the protein is folded (3D structure)

Gel filtration

is a chromatography technique used to separate molecules based on size. It allows larger molecules to pass through while smaller ones are retained in the gel matrix, enabling the separation of proteins, nucleic acids, and other biomolecules.

To further separate the protein of interest from other proteins, which is the most laborious part of the protein purification process, various types of _______ are used. These procedures take advantage of the difference in the proteins’ size, charge, and binding affinity to some ligands

Chromatography

Ion exchange chromatography

separates proteins according to their charge

• If the protein of interest has overall negative charge, a resin that contains positively charged groups is used

• To retain positively charged proteins, a negatively charged resin is employed.

• proteins with lower charge move through column faster + have higher affinity to resin, require higher salt concentration to be eluted from resin

what are the most commonly used resin for exchange chromatography?

anion → Q-resin, DEAE-resin

cation → S-resin, CM resin

• resins are chosen based on the charge of the protein

Affinity Chromotography

Based on the ability of the protein of interest to bind specific ligands with high affinity

The target molecules become entrapped on a stationary phase, while other proteins, which do not possess the affinity to the immobilized ligand, freely pass through the column.

The entrapped protein of interest is then recovered from the immobilized ligand using a high salt concentration, buffers with low pH, or a denaturing agent (e.g., 8 M urea). The effluent is removed by dialysis.

immobilized metal affinity chromatography (IMAC)

A technique that uses metal ions immobilized on a resin to selectively bind proteins that have affinity for these metals. Commonly utilized for purifying histidine-tagged proteins.

A commonly used approach involves introduction of 6–8 histidine residues into the C-terminal fragment of the protein by means of genetic engineering prior to the cloning of the protein gene in a bacterial or yeast culture.

The polyhistidine tag of the protein has an affinity for such metal ions as nickel and cobalt. The protein containing the tag is retained in the column with immobilized nickel ions, while all untagged proteins freely pass through the column.

Beer Lambert Law

A=ε×C×l

where A is absorbance, ε is the molar absorptivity, C is the concentration of the solution, and l is the path length of the light through the sample.

if nucleic acid contamination of the protein sample is possible, to estimate the protein concentration in the sample the absorbance at both 280 and 260 nm is taken into account using the following formula:

C(mg/ml) = 1.55 x A₂₈₀ - 0.76 x A₂₆₀

Bradford Essay

A method for determining protein concentration via color change, resulting from the binding of Coomassie Brilliant Blue dye to proteins. The intensity of the color is measured spectrophotometrically

To enable proteins to have identical charge per a mass unit and to fractionate them by size only, a technique of denaturing electrophoresis called ______________________ is employed.

sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Gel Electrophoresis

• For protein electrophoresis mostly polyacrylamide gels are used

• Positively charged proteins will migrate from the positively charged electrode (anode) to the negatively charged electrode (cathode) in the electric field. Negatively charged proteins (e.g., pepsin) will migrate in the opposite direction.

R_f

which is determined as the distance travelled by a protein divided by the gel length, has a linear correlation with the logarithm of its molecular mass.

avg mw of an amino acid

110 Da

#of amino acids in a protein

MW of protein/110 Da

capillary electrophoresis

carried out in a very thin tube filled with electrolytes

• fast and high efficiency due to high voltages

• both positive and negative ion travel though the capillary drive by electroosmotic fluid, typically directed toward (-) charged cathode

• deteteced ions are peaks on the electophoreogram.

isoelectric focusing (IEF)

is a technique for separating proteins based on their isoelectric point (pI). It involves creating a pH gradient in a gel or solution, causing proteins to migrate until they reach the pH where their net charge is zero.

2D gel electrophoresis

• Combines IEF and SDS-PAGE

• 2 stages of protein separation

• IEF first, then gel tube (contains proteins separated by pI) is placed on top o f an SDS-containing polyacrylamide gel slab, and the proteins are fractionated by size

  • for large-scale studies of proteins

Edman’s degradation

• amino acids are sequentially cleaved off from the peptide starting from the N-terminus

• done by PITC or Edman’s reagant w/ an uncharged terminal amino group

• PTC derivative → thiazoline derivative → PTH derivative

• peptide sequenators accelerate the procedure

• lager proteins must be cleaved into smaller fragments before the ED