Structural Biochemistry/Final Review/Midterm II

Quick Comparisons

1. D configuration is much more common than L isomer in sugars
2. Unsaturated and shorter fatty acid chains tend to be more fluid and melt at a lower temperature
3. Bilayer is preferred for lipids in water over a micelle
4. Integral proteins span membranes and are hard to remove compared to peripheral proteins which only interact via electrostatics/hydrogen bonding
5. Alpha helix proteins tend to span membrane while beta strands tend to form channels
6. Adenine and guanine are two ringed purines (Guanine has an extra carbonyl group)
7. Cytosine and thymine are one ringed pyrimidines (Thymine has an extra carbonyl group)
8. Adenine and thymine form two bonds while cytosine and guanine forms three
9. Difference between thymine and uracil is that thymine has a CH3 substitutient
10. A nucleotide is a nucleoside joined to one or more phosphates with ester bonds, such as ATP

What types of bonds join these molecules together?

1. Sugars in a disaccharide- glycosidic linkage
2. Backbone of DNA- phosphodiester linkage
3. Glycerol and fatty acids in a lipid- ester linkage

Name the main types of sugars and give an example

1. Aldose: D-Glucose
2. Ketose: D-Fructose
3. Pentose: A-D-Fructofuranose
4. Hexose: A-D-Fructopyranose
5. Reducing Sugars: Glucose, galactose and fructose}}

Name the three common disaccharides

1. Sucrose: Glucose and fructose
2. Lactose: Glucose and galactose
3. Maltose: Glucose and glucose

Name the most common polysaccharides

1. Glycogen: Branched A linked glucose, animal energy storage
2. Amylose (starch): Unbranched A linked glucose, plant energy storage
3. Amylopectin (starch): Branched A link glucose, plant energy storage
4. Cellulose: A linked for energy storage and B linked straight chain for structure

Name common features shared by biological membranes

1. Sheet like structures only two molecules thick
2. Consists mainly of lipids and proteins
3. Lipids have hydrophilic and hydrophobic ends and form bilayers
4. Specific proteins mediate distinctive functions
5. Membranes are noncovalent assemblies held by noncovalent interactions
6. Membranes are asymmetric- inside and outside surface distinct
7. Membranes are fluid- molecules can move around easily but hard to rotate
8. Most membranes are electrically polarized such that the inside is negative

Describe the three common types of membrane lipids

1. Phospholipids with hydrophobic fatty acid tail and hydrophilic phosphate/alcohol head
2. Glycolipids with hydrophobic fatty acid tails and hydrophilic sugar heads usually of glucose or galactose
3. Cholesterol- steroid derived liquid with hydrocarbon tail and hydroxyl group; rarely found in prokaryotes but common in animal membranes

Describe the main differences between archea membranes and eukaryote/bacterial ones

1. Nonpolar chains are joined to glycerol by ether rather than ester link since it is more resistant to hydrolysis
2. Alkyl chains are branched rather than linear which resist oxidation
3. Stereochemistry of central glycerol is inverted in archea membranes

Identify some key features of the fluid mosaic model

1. Molecules can move laterally with ease but it is difficult to rotate
2. Lack of rotation allows membrane to remain asymmetrical
3. Bacteria regulate fluidity by varying degree of unsaturation and length of fatty acid chains
4. Animals regulate fluidity by cholesterol concentration, which is bulky and disrupts movement

Identify some membrane bound organelle within the cell

1. Nucleus
2. Endoplasmic reticulum (ER)
3. Mitochondria

Describe the Avery-MacLeod-McCarty experiment with bacteria and mice

Live virulent bacteria are shown to kill mice while a similar but nonvirulent strain does not. Killing the virulent strain and then injecting the mice does not harm the mice. Killing the virulent strain and then either adding its DNA or the dead strain itself to the nonvirulent strain caused a transformation. The newly transformed nonvirulent strain is now virulent and will kill the mice. This showed that DNA contains genetic information.

Describe the Hershey-Chase experiment with radio marked viruses

Hershey and Chase added a radioactive marker to two viruses: one set had markers on its protein coat and the other had markers on its DNA. The viruses were allowed to infect bacteria and in the end, only the bacteria infected by the virus with radioactive DNA were radioactive. Thus, virus only transferred DNA to bacteria meaning that DNA must be the genetic code.

Name three main units of nucleic acids

1. Sugar- either deoxyribose or ribose
2. Base- nitrogenous base attached to the sugar
3. Phosphate- attached in between sugar molecules in the backbone

What are the main features of Watson and Crick’s double helix model of DNA

1. Two polynucleotide chains around a common helical axis running in opposite directions
2. Sugar-phosphate backbone on the outside and nitrogenous bases on the inside
3. Bases are perpendicular to axis, separated by 3.4 angstrom, in a pattern that repeats every 10 bases; there is a rotation of 36 degrees per base
4. The diameter of the helix is 20 angstroms

What are the forces that stabilize DNA?

1. Stacking interactions between stack bases are weak and thus more stable
2. Hydrogen bonds between bases is strong and holds helix together
3. Hydrophobic effect of burying bases in the interior makes it more stable
4. Charge-charge interactions such as the repulsion of phosphate in the backbone is reduced by the presence of cationic proteins such as lysine and arginine

NOTE: DNA can coil further into secondary and tertiary structures via super coiling (Ex: circular)

Describe the significance of DNA melting

DNA can be reversibly melted because at the melting temperature, the chains do not break down but the double helix does separate into two strands. This results in an increase in UV absorption since the double helix absorbs less due to the hypochromic effect. Once the temperature dips below the melting point, the double helix can be reassociated in a process called annealing.

What are four requirements regarding the ability of DNA polymerase to catalyze phosphodiester bond formation?

1. The reaction requires all four activated precursors- dATP, dGTP, dCTP and dTTP as well as Mg ion
2. The new DNA chain is assemble directly on a pre-existing template
3. A primer must be present to begin synthesis; this primer is a free 3’-OH and elongation of DNA proceeds in the 5’ to 3’ direction
4. DNA polymerase can remove mismatched nucleotides and correct mistakes

NOTE: Meselson-Stahl experiment demonstrated that DNA replicated in a semi-conservative fashion, meaning that half of the newly synthesized strand comes from parental DNA. The experiment uses radioactive nitrogen markers and measured the radioactivity of parent and daughter strands.

Explain how retroviruses attack cells

The retrovirus carries two single-stranded RNA molecules. Once it enters the cell, an enzyme known as reverse transcriptase is used to form a DNA complementary to RNA. The RNA is then dissolved and what remains is a DNA transcript of the original viral RNA, which the cell then makes into a double helical viral DNA strand. This strand is incorporated and the cell is infected.

Identify some important forms of RNA

1. Messenger RNA- template for protein synthesis or translation
2. Transfer RNA- Carries amino acid to ribosomes for peptide formation
3. Ribosomal RNA- Main component of ribosomes, both as structure and as enzyme
4. Small nuclear RNA- Splices RNA exons
5. Signal recognition particle- RNA-protein complex that targets new proteins to right places
6. Micro RNA- Inhibits translation
7. Small interfering RNA- Promotes messenger RNA degradation
8. RNA telomerase- An enzyme that maintains chromosomal telomeres during replication

What are the key features of RNA polymerase?

1. Requires a template, usually double stranded DNA but sometimes single stranded DNA
2. Four activated precursors- ATP, GTP, UTP and CTP
3. A divalent metal ion such as Mg or Mn
4. Elongates in 5’ to 3’ direction
5. Cannot correct error

What are the key differences between DNA and RNA polymerase?

1. DNA can correct errors but RNA cannot
2. DNA needs primer but RNA does not
3. DNA needs dTTP precursor while RNA needs UTP

What are the key parts of the genetic code, the relation of DNA bases to protein peptides?

1. Three nucleotides form a codon which encodes an amino acid
2. The code does not overlap, meaning each nucleotide participates in only one set of three nucleotides that form a codon
3. The code has no punctuation, meaning the codons code for amino acids continuously, and all bases are part of a codon
4. The genetic sequence is degenerate meaning there are many codons for each amino acid
5. The genetic code is universal among living organisms

NOTE: Methanonine is the start codon and always start a peptide sequence, while stop codons are read by release factors rather than transfer RNA anti-codons.

What are introns and exons? How does the split gene sequence benefit an organism?
1. Introns are noncoding DNA that do not call for proteins and exist only in eukaryotic cells
2. Exons are DNA that does code for proteins and is copied during transcription
3. Introns are spliced out prior to DNA transcription at GU/AG which signal splice sites
4. Split genes allows for exons shuffling which allows exons to be rearranged quickly and efficiently. Since exons code for discrete functional and structural units, this allows the structure to be changed easily.
5. Split genes also allows the generation of a series of related proteins by splicing the same RNA chain in difference ways

What are the key tools in the analysis of genes?
1. Restriction enzymes- Enzymes that cut genes at precise locations
2. Blotting techniques- Southern blot separates and characterizes DNA while Northern does the same for RNA
3. DNA sequencing- Determines the precise nucleotide sequence in DNA
4. Solid-phase synthesis- Synthesizes precise sequences of DNA and uses it to identify others
5. Polymerase chain reaction- Amplifies a segment of DNA that makes it easier to characterize and manipulate genes

Techniques in DNA synthesis and manipulation

Restriction enzymes
1. Restriction enzymes cleave DNA into specific fragments by recognizing certain sequences and using them as cleave sites
2. The results can then be separated using gel electrophoresis
3. The resulting fragments can be identified by hybridizing it with labeled complementary DNA strand using the Southern blot

Southern blot
1. A mixture of restriction fragments are separated via electrophoresis on agarose, denatured to form single strands and transferred onto a nitrocellulose sheet
2. The positions of the fragments are preserved and exposed to a radioactive phosphorus labeled, single stranded DNA probe.
3. The DNA probe will hybridize with a fragment having the complementary sequence and the radioactive marker will identify which fragment was the match
4. A similar technique for analyzing RNA is known as the northern blot.
5. From earlier chapters, the method for detecting protein with stained antibodies was known as the western blot

DNA Sequencing by controlled termination of replication
1. Chain termination DNA sequencing generates fragments whose length depends upon the last base in the sequence
2. The same procedure is preformed on four reactions at once.
3. A DNA polymerase is used to make the complement of a sequence and is primed by a synthesized fragment complementary to part of the sequence.
4. An analog of one of the nucleotide is used in synthesis and when it is used, it terminates the chain.
5. By using an analog for a different nucleotide each time, we can determine the sequences by analyzing the resulting fragments.

DNA Solid Phase Synthesis
1. Activated monomers are protonated molecules that are added to a growing chain and form a phosphate triester bond
2. The triester is oxidized by iodine
3. The DMT protecting group is removed by reacting it with the acid and this elongates the chain by 1 unit
4. Takes about 10 minutes to elongate a chain

Polymerase Chain Reaction (PCR)
1. Can be used on targets whose flanking sequences are known
2. The parent DNA is headed to excess primer to separate the strands
3. The solution is then abruptly cooled to allow the primers to hybridized to a DNA strand
4. DNA synthesis begins as DNA polymerase is added
5. The chain is copied from both primers producing two new double strands
6. The two new double strands are separated by heat and the cycle starts again
7. The quantity grows exponentially and can easily amplify DNA a billion times
8. Materials required includes the appropriate DNTP and the usual metal Mg, along with buffer and constant temperature change

Recombinant DNA technology
1. A plasmid is a circular DNA that has our gene of interest and an origin of replication
2. Restriction enzymes can be used to cut a particular piece of DNA; cut fragments can then be annealed together with DNA ligase
3. A similar method can be used to add a sequence to a gene though it is much faster to do this using PCR
4. DNA cloning in bacteria is usually done with plasmids or lambda phage
5. A cloning vector is cleaved with an restriction enzyme, a DNA fragment of interest is obtained by cleaving and the two fragments are ligated together. The recombinant DNA is then introduced into the host cell which begins to make many copies of the recombinant DNA.

What are the two kinds of homologs and give an example of each?

A paralog is a homolog (similar protein or gene) from the same species but with different function such as two different enzymes in a human.

Orthologs are from different species but perform a similar function such as digestive enzymes in humans and cows.

What is sequence alignment and how does it work?

Sequence alignment is a method to detect homologous genes or proteins. It is typically done on amino acid sequences because it is more sensitive as there are more amino acids than nitrogenous bases. To do the alignment, one sequence is slid over another and gaps are introduced to account for deleted amino acids. To know if an alignment is meaningful or not, shuffle the original sequence into a random sequence and then run an alignment with the random sequences and compare the scores. The random sequence should have a much lower score relative to the authentic sequence.

What are substitution and identity matrices? How are they used?

A substitution matrix allows us to account for the type of substitution, whether or not amino acids are swapped for similar ones or dissimilar ones. The deduced matrices can help us identify amino acids that are conserved more than the others. This has a higher level of sensitivity because it accounts for minor (conservative) substitutions that are relatively irrelevant to the central structure such as alanine for glycine. The identity matrix method is similar to sequence alignment because it only considers the identity of each amino acid. The lack of statistical similarity does not rule out homology as divergent evolution may have changed the proteins too much for a simple comparison.

What is structural alignment and how is it used?

Structural alignment refers to the conservation of tertiary structures in proteins. Tertiary structures in proteins tend to be more conserved than the primary structure of amino acid sequences. For example, the heme group is conserved in both hemoglobin and myoglobin. Also, proteins with different amino acid sequences may have similar three dimensional structures. It can be used to evaluate the effects of sequence alignments by generating a template which shows conserved residues and their functionality. Repeated motifs can be detected by aligning sequences with themselves.

What is divergent and convergent evolution and give a biochemical?

Divergent evolution is the process by which living things differentiate from a common ancestor, an example is one protein changing into multiple forms.

Convergent evolution is the natural evolution of two independently evolving things towards each other, an example is how two proteases evolve nearly identical active sites despite differing tertiary structures.

Centrifugation

- Mixture of particles that need to be separated are placed onto the surface of a vertical column of liquid.

- The density of which progressively increases from top to bottom → centrifuged.

- Although the particles in suspension are individually denser than the liquid at the top of the gradient, the average density for the sample is lower; only under such conditions could the sample zone be supported by the top of the density gradient.

Mass Spectroscopy (MALDI-TOF)

- Characterization and identification of protein better than SDS-PAGE

- MADLI-TOF has higher sensitivity and can separate small protein samples

STEPS:

1. Sample protein is separated by 2-D gel

2. Specific cleavage (like trypsin) cuts the proteins

3. Separated peptides are identified by MALDI-TOF

- Heavy peptides will move slowly, and lighter peptides will move faster

4. As peptides move along the tube, the different peptides take different amounts of time to reach to the end of the tube

- These separated peptides can be analyzed by matching with the computer simulated database

ADVANTAGES:

• Robustness

• High Speed

• Immunity to contaminants, biochemical buffers, and common additives

Peptide Sequencing

- Find out amino acid composition

- By using indicator dye to quantify-ninhydrin or fluorescamine

- Sequence one AA at a time

- By using Edman Sequencing : process of purifying protein by removing one residue at a time from the amino end of a peptide

Peptide Synthesis

• Make antibodies

– Peptides can act as antigens, which will stimulate the immune system of the body to produce antibodies to target the peptide. These antibodies can be used to isolate a protein

• Isolate receptors for hormones of other signaling molecules

• Work as drugs

– Synthetic peptides can be used as drugs.

– Example: Vasopressin, which is used to treat patients with diabetes

• Understand protein folding

– Extremely helpful because synthetic peptides can include many AA’s that are not found in normal proteins; meaning these peptides are not limited to the standard 20 AA’s → resulting in a greater variety of structures

Procedure:

• 1. Attach protected AA

• 2. Deprotect amino terminal

• 3. Coupling to the second protect AA

• 4. Deptrotect amino terminal

• 5. Disconnect dipeptide

X-Ray Crystallography

• Can reveal 3-D structures of thousands of proteins

• 3 Components – Protein Crystal, Source of X-Rays, Detector

o Answers to the level of individual residues and atoms

Electron Microscopy

• Can reconstruct 2D images of a molecule to 3D

Requirements:

1. Reasonable size of proteins without need to use crystals (samples don’t have to be pure and only takes very little sample)

2. Molecules must exist in many identical copies

Steps:

1. Sample Preparation

- Sample is collected and placed on a metal plate to generate the best contrast

- Negative Staining

• Molecules are absorbed to a continuous carbon film in which molecules are put into a metal plate by drying

2. Particle Picking

o Particles have to be separated and classified according to their similarities

3. Generate Initial Model

– Random Conical Tilt Reconstruction

• reconstruction of the 3D images; one at high tilt angle, and the other at untilt angle. Tilt angles allow the testing samples to align in unique orientations

4. Refinement

- From refined data, a new 3D structure of a molecule is reconstructed

Nucleic Acids

Three main chemical units of nucleic acids:

1. Nitrogenous base

2. Sugar

3. Phosphate group

Nucleotides

- Units of DNA
- consists of sugar (ribose, deoxyribose), phosphate group, and a base (A,G,T,C)
- Joined by 3’-5’ Phosphodiester bonds

Nucleosides

- Units of RNA
- Consists of sugar (ribose, deoxyribose) and a base (A,G,T,C)

DNA

Stores genetic information
Codon – sequence of three bases, responsible for encoding of a single AA

– PROPERTIES:

1. 2 strands (anti-parallel and complementary)
2. Made up of deoxyribose sugar, phosphate backbone, and nucleic acid base
3. Bases are perpendicular to the helix axis (3.4A)
4. Strands held together by H-bonds to form double helix (A=T, G=C)
5. Direction: 5’  3’
6. Repeats every 10 bases
7. Weak forces stabilize DNA because of hydrophobic effects and Van Der Waals
8. DNA chain = 20A wide

STRUCTURE:

- A and B = Right handed
- Z = Left handed

cDNA LIBRARY:

- Library of mRNAs: made up of introns and exons and is able to isolate genes

Flow Of Genetic Information

- Replication: DNA to DNA
- Transcription: DNA to RNA
- Translation: RNA to Proteins

Hypochromic Effect

- Describes the decreasing absorbance of UV when forming a double strand
- As you increase the temperature on a DNA double strand it becomes denatured and is therefore a single strand, leaving the bases more exposed and more able to absorb UV.

Additional Notes

Notes on RNA:

RNA sequences can be compared in a similar manner which will reveal important information about the secondary structures of RNA.

Notes on evolutionary trees:

The alignment data can be used to generate an evolutionary tree, such as one for the globins which shows that hemoglobin B was the most recent development.

Notes on modern technology:

Ancient DNA can sometimes be amplified and sequenced which allows us to compare what species are more closely related on the evolutionary tree. This has for example revealed that Neanderthals are a branch of modern humans that became extinct, as opposed to a step along the evolutionary line. Molecular evolution can also be examined experimentally by using combinatorial chemistry. This is done by creating a large random pool of molecules and selecting for a specific function.