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Notes for Lectures 20&21

by: Annie Notetaker

Notes for Lectures 20&21 BCM 475 - M001

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Biochemistry I
M. Braiman, R. Welch
Class Notes
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This 10 page Class Notes was uploaded by Annie Notetaker on Wednesday November 4, 2015. The Class Notes belongs to BCM 475 - M001 at Syracuse University taught by M. Braiman, R. Welch in Fall 2015. Since its upload, it has received 30 views. For similar materials see Biochemistry I in Biochemistry at Syracuse University.


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Date Created: 11/04/15
Lecture 10/19: Evolution and Bioinformatics (p. 173-191) Structure and Function • Bovine ribonuclease and human ribonuclease are structurally and functionally similar -Angiogenin possesses a structure similar to that of ribonuclease—indicating a possible evolutionary relationship (human ribonuclease and human angiogenin are paralogs) Sequence Comparison Methods • Door to understanding the function and mechanism of newly obtained sequences • Utilized for the determination of evolutionary pathways Homology • Homologous molecules (homologs) come from a common ancestor • Homologs: 1. Paralogs 2. Orthologs -Paralogs: “homologs that are present within one species” but “differ in their detailed biochemical functions” -Orthologs: “homologs that are present within different species and have very similar or identical functions” • “Understanding the homology between molecules can reveal the evolutionary history of the molecules as well as information about their function” Detecting Homology • Similarity in amino acid sequences ▯ common ancestor ▯ similar structure ▯ similar functions ▯ similar mechanisms • Globins -Class of proteins including myoglobin and hemoglobin -Sequence alignment method utilized to detect common sequences in hemoglobin and myoglobin Comparing the Amino Acid Sequences of ????-Hemoglobin and Myoglobin • Sequence alignment 1. Slide amino acid sequences of hemoglobin and myoglobin past each other, one amino acid at a time 2. “Count the number of matched residues, or sequence identities” - ????-Hemoglobin and myoglobin: 23 sequence identities 3. Analyze graph plotting number of matches vs. alignment • Sequence alignment techniques must take into consideration insertions or deletions that may have occurred -By shifting one sequence of comparison by a certain number of residues and subsequently comparing to an adjacent sequence, one can detect different amino acid identities (matched residues) -“By introducing a gap into one of the sequences, the identities found in both alignments will be represented” 1 • Alignment with gap insertion -The greater number of gaps, the increased number of sequence identities -Because an excessive number of gaps can lead to an unreasonably high number of matched amino acid residues, mathematical methods have been developed to compensate for such cases -E.g. a scoring system that counts each identity as +10 points and each gap as -25 points Determining the Statistical Significance of Sequence Alignments • Not all sequence alignments correlate to homology -“It is the order of the residues within their sequences that implies a (evolutionary) relationship between them” • Steps for determining the statistical significance of sequence alignments: 1. Shuffle, or randomly rearrange one of the two sequences of comparison 2. Repeat sequence alignment 3. Calculate a new alignment score for shuffled sequence 4. Repeat process 5. Create a histogram depicting the number of alignments for many shuffled sequences plotted against the alignment score -Unshuffled sequences with a significantly greater alignment score than those of other shuffled sequences indicate the statistical significance of the sequence alignments -E.g. “The alignment score for unshuffled alpha-hemoglobin and myoglobin is substantially greater than any of these scores (for shuffled sequences), strongly suggesting that the sequence similarity is significant” and hence that alpha- hemoglobin and myoglobin are homologous Detecting Distant Evolutionary Relationships • Substitution matrices enable the detection of evolutionary relationships between proteins that have undergone amino acid substitutions • Substitution types: 1. Conservative substitution -A substitution in which one amino acid is replaced “with another that is similar in size and chemical properties” -No significant effect on protein structure or function -Common amino acid change 2. Nonconservative substitution -A substitution in which “an amino acid is replaced by one that is structurally dissimilar” 3. Single-nucleotide substitution -Common amino acid change 2 • Steps for comparing sequences with adjustments for substitutions that may have occurred: 1. “Examine the substitutions that have actually taken place in evolutionarily related proteins” 2. Derive a substitution matrix from data obtained in step one • Substitution matrix -“A substitution matrix describes a scoring system for the replacement of any amino acid with each of the other 19 amino acids” -Large positive score in substitution matrix ▯ common amino acid substitution -Large negative score in substitution matrix ▯ rare amino acid substitution -E.g. Blosum-62 (Look at Figure 6.9) • Blosum-62 -Substitution matrix -Depicts conservative and nonconservative substitutions with positive and negative scores respectively -“This scoring system detects homology between less obviously related sequences with greater sensitivity than would a comparison of identities only” Alignment of Identities Only Versus the Blosum-62 • To detect homology between leghemoglobin and human myoglobin, two different scoring systems were used: 1. Identity-based scoring system and 2. Blosum-62 - According to the identity-based scoring system, there is a 1 in 20 chance of no statistical significance existing for the aligned sequences - According to Blosum-62, there is a 1 in 300 chance of no statistical significance existing for the aligned sequences - Blosum-62 strengthens the conclusion that homology exists between leghemoglobin and human myoglobin Rules of Thumb for Sequence Analysis • Sequence identities greater than 25% ▯ most likely homologous sequences • Sequence identities less than 15% ▯ “alignment alone is unlikely to indicate statistically significant similarity” • Sequence identities between 15 and 25% ▯ requires further analysis • Note, “the lack of a statistically significant degree of sequence similarity does not rule out homology” Databases • Searching databases for homologous sequences is often the first step performed in identifying a newly obtained protein sequence • The Basic Local Alignment Search Tool (BLAST) is used to align newly obtained amino acid sequences with other known sequences -Investigators in 1995 “were able to identify likely functions for more than half the proteins within this (bacterium Haemophilus influenzae) organism solely by sequence comparisons” via BLAST 3 Three-Dimensional Structure and Homology • “Because three-dimensional structure is much more closely associated with function than is sequence, tertiary structure is more evolutionarily conserved than is primary structure” -E.g. While the tertiary structures of hemoglobin, myoglobin, and leghemoglobin are conserved (each possess a heme group that binds oxygen), this conservation is not apparent in the primary structures of the globins -E.g. Actin and heat shock protein 70 (Hsp-70) “were found to be noticeably similar in structure (three-dimensional) despite only 15.6% sequence identity” • Three-dimensional structure can be used to evaluate sequence alignments by the following steps: 1. Identify highly conserved, functionally significant residues within related proteins -E.g. identify the histidine residue important for the heme group’s function that is conserved in hemoglobin, myoglobin, and leghemoglobin 2. Generate a sequence template with the highly conserved key residues -Sequence template: “a map of conserved residues that are structurally and functionally important and are characteristic of particular families of proteins” -By generating a sequence template, one can “recognize new family members that might be undetectable by other means” Sequence Alignment of Internal Repeats • “Repeated motifs can be detected by aligning sequences with themselves” Convergent Evolution • “The process by which very different evolutionary pathways lead to the same solution” • E.g. chymotrypsin vs. subtilisin -Both enzymes possess a catalytic triad in their active sites -The enzymes, however, are not homologous -Chymotrypsin is structurally composed mostly of beta sheets while subtilisin is composed of many alpha-helices, making it unlikely that these two proteins are homologous Comparison of RNA Sequences • Comparison of homologous RNA sequences can serve as a starting point for identifying homology and RNA secondary structures • E.g. “a comparison of (RNA) sequences in a part of ribosomal RNA taken from a variety of species” - Figure 6.20 - For each of the different sequences compared, “the bases in positions 9 and 22, as well as several of the neighboring positions, retain the ability to form Watson- Crick base pairs even though the identities of the bases in these positions vary” 4 - The comparison of the RNA sequences enabled the identification of the possible RNA secondary structure Evolutionary Trees • Figure 6.21 • Constructed from aligned sequences • In evolutionary trees, “the length of the branch connecting each pair of proteins is proportional to the number of amino acid differences between the sequences” • “Evolutionary trees can be calibrated by comparing the deduced branch points with divergence times determined from the fossil record” -Fossil records enable scientists to estimate the time of evolutionary divergence -E.g. an evolutionary tree for globin depicts how “the duplication leading to the two chains of hemoglobin” occurred 350 millions years ago while fossil records indicate how “the jawless fish such as the lamprey, which diverged from bony fish approximately 400 millions years ago, contain hemoglobin built from a single type of subunit” Modern Techniques Utilized to Understand Molecular Evolution • Biochemistry techniques used to understand evolution: 1. Polymerase chain reaction (PCR) and 2. Combinatorial chemistry • PCR -Utilized to amplify ancient DNA for sequencing -Was used to amplify mitochondrial DNA from a Neanderthal fossil -“Investigators have completely sequenced the mitochondrial genome” from the Neanderthal and comparison of the DNA sequences of Neanderthal with those from Homo sapiens via an evolutionary tree revealed that the “Neanderthal is not on the line of direct descent leading to Homo sapiens but, instead, branched off earlier and then became extinct” Evolution in the Laboratory • Essential processes of evolution: 1. “The generation of a diverse population” 2. “The selection of members based on some criterion of fitness” 3. “Reproduction to enrich the population in these more-fit members” • Nucleic acids can undergo all three processes mentioned above and combinatorial chemistry can be used to synthesize nucleic acid molecules • Before proteins existed, did RNA molecules function as catalysts? -To answer, scientists performed the following experiment: 1. A randomized pool of RNA molecules was synthesized by combinatorial chemistry 2. The newly synthesized RNA molecules were passed through an ATP affinity column for the selection of ATP-binding molecules 3. The ATP affinity column was washed with excess ATP 4. ATP-binding RNA molecules were eluted off the column 5. The ATP-binding RNA molecules “were allowed to replicate by reverse transcription into DNA, amplification by PCR, and transcription back into RNA” 5 6. “The new population was subjected to additional rounds of selection for ATP- binding activity” 7. Replication and selection repeated 8. Final RNA products that bound ATP with high affinity were isolated 9. Final RNA products were characterized - NMR revealed how RNA forms a pocket in which ATP can fit and bind (an evolved ATP-binding RNA molecule) Lecture 10/23: Genetic Information I (p. 109-121) Gene Expression Transcription T anslation • DNA ▯ RNA ▯ Protein Nucleic Acids • Linear polymers -Monomer unit within polymer▯ nucleotide -One nucleotide consists of the following: 1. A sugar 2. A phosphate 3. One of four bases • E.g. deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) Nucleic Acid Structure • Sugar-phosphate backbone -Structural support for nucleic acids - Sugars linked by phosphodiester bridges -“3’ –hydroxyl group of the sugar moiety of one nucleotide is esterified to a phosphate group, which is in turn, joined to the 5’- hydroxyl group of the adjacent sugar” -Each phosphodiester bridge is negatively charged and therefore less prone to hydrolytic attack than other esters (“this resistance is crucial for maintaining the integrity of information stored in nucleic acids”) -A polynucleotide backbone contains 6 rotatable single bonds per unit • Bases -Carry genetic information -Adenine (A) ▯ purine -Guanine (G) ▯ purine -Cytosine (C) ▯ pyrimidine -Thymine (T) ▯ pyrimidine -Uracil (U) ▯ pyrimidine RNA vs. DNA • DNA -Sugar ▯ deoxyribose -2’ carbon atom of sugar lacks oxygen (2’ carbon atom has two hydrogen atoms) 6 -The absence of a hydroxyl group at the 2’ position of DNA enables this nucleic acid to be less susceptible to hydrolytic attack and hence more stable in carrying genetic information than RNA -Bases: A—T and C—G • RNA -Sugar ▯ ribose -2’ carbon atom of sugar contains a hydroxyl group -Bases: A—U and C—G Nucleoside • Base – sugar • Non-phosphorylated • DNA nucleoside units: 1. Deoxyadenosine 2. Deoxyguanosine 3. Deoxycytidine 4. Thymidine • RNA nucleoside units: 1. Adenosine 2. Guanosine 3. Cytidine 4. Uridine • “N-9 of a purine or N-1 of a pyrimidine is attached to C-1’ of the sugar by an N- glycosidic linkage” (the ????-glycosidic linkage with a base is above the plane of the sugar) • The glycosyl bond (glycosidic bond) in a nucleoside can rotate 180 degrees to go from an anti conformation to a syn conformation and vice versa Nucleotide • “Nucleoside joined to one or more phosphoryl groups by an ester linkage” • Nucleotide triphosphates -Nucleosides bound to three phosphoryl groups -Monomers of DNA and RNA • 5’ –nucleotide -Nucleoside 5’ –phosphate -Compound with a phosphoryl group bound to C-5’ of sugar -E.g. adenosine 5’ –triphosphate (ATP) Structure of a DNA Chain • DNA is conventionally written from the 5’ end to the 3’ end • Polarity exists in a DNA chain -Free 5’- OH group -Free 3’- OH group DNA Length • DNA molecules are very long polymers that are tightly compacted in cells • Figure 4.8 lysed E. coli cell • E.g. human DNA has a total contour length of greater than 1 meter; the average contour length of the 46 chromosomal DNA molecules is greater than 2 cm Watson-Crick Model of Double-Helical DNA • The double-helix facilitates DNA replication 7 • Features: -“Two helical polynucleotide chains are coiled around a common axis with a right- handed screw sense” -Antiparallel chains with opposing directionality -The hydrophilic sugar-phosphate backbone is located on the exterior of the double helix while the hydrophobic bases are located inside the helical structure -“The bases are nearly perpendicular to the helix axis” in B-DNA -“Adjacent bases are separated by 3.4 angstroms” -About 10 bases per turn of right-handed helix of B-DNA -Diameter of the double helix: 20 angstroms Watson-Crick Base Pairs • Base pairs bind via hydrogen bonding • For DNA: A—T and C—G • For RNA: A—U and C—G • Chargaff’s rule G/C = A/T = 1 Contributions to the Stability of the Double-Helical Structure • Base stacking -The stacking of base pairs in a double-helical structure leads to the stability of the helix via: 1. The hydrophobic effect -Hydrophilic sugar-phosphate backbone located on outside of helix is exposed to water while hydrophobic bases are protected from an aqueous environment by lying inside the double helix 2. Van der Waals forces -These forces are present between stacked base pairs and further stabilize a double helix -Due to correlated motion of pi electrons • Hydrogen bonds -Responsible for holding base pairs together -Although the hydrogen bonds holding base pairs together are fairly weak, they “stabilize the helix because of their large numbers in a DNA (or RNA) molecule” • Electrostatic interactions -“Mutual repulsion of negatively charged phosphate groups is relieved by having the phosphates on the exterior of the double helix” • Counterion condensation -“Occurs between adjacent pairs of phosphates on the same chain; each cation binding site is occupied 80% in the presence of 150 mM Na+ to 98% in the presence of physiological concentration of Mg2+” Double-Stranded DNA has 10 Distinct Stacking Interactions • Base-stacking is a more significant contributor to the stability of the double-helical structure (0.5-2 kcal/mol) than hydrogen bonding 8 -The stacking of C and G base pairs is more favorable than the stacking of A and T base pairs and the base-stacking of C-G is more significant than the three hydrogen bonds between C and G base pairs (two hydrogen bonds connect A and T base pairs) Structural Forms of DNA • A-form helix -Form for double-stranded RNA -Form of dehydrated DNA -Right-handed double helix -Anti-parallel strands -Shape: broadest helix type -Shorter than B-DNA -Anti glycosidic bond -Tilt of base pairs from perpendicular to helix axis: 19 degrees • B-form helix -Most common form for DNA -Right-handed double helix -Anti-parallel strands -Shape: Intermediate helix type -Narrower and longer than A-DNA -Anti glycosidic bond -Tilt of base pairs from perpendicular to helix axis: 1 degree Biochemical Basis for the Structural Differences Between the A and B Form of DNA • Different sugar puckers A-form ▯ “C-3’ carbon atom lies above the approximate plane defined by the four other sugar nonhydrogen atoms (conformation called C-3’ endo)” B-form ▯ “each deoxyribose is in a C-2’ –endo conformation, in which C-2’ lies out of the plane” Z-DNA • Another helix type in addition to A and B • Shape: narrowest helix type • Left-handed double helix • Zigzagged phosphates in backbone • Alternating anti and syn glycosidic bonds Different DNA Structures • Circular DNA • Supercoiled DNA -Compact DNA -“Supercoiling may hinder or favor the capacity of the double helix to unwind and thereby affect the interactions between DNA and other molecules” 9 Structures of Single-Stranded Nucleic Acids • E.g. stem-loop structure “created when two complementary sequences within a single strand (of DNA or RNA) come together to form double-helical structures” The Double Helix and the Transfer of Genetic Information • Because a particular base on one strand of DNA is always paired with a specific base on an adjacent strand, “the sequence of bases of one strand of the double helix precisely determines the sequence of the other strand” • DNA is replicated via semiconservative replication The Melting and Annealing of the Double Helix • A disruption of the hydrogen bonds holding DNA base pairs together will lead to the separation of the double helix into its two component strands • Melting is “the dissociation of the double helix” -Melting temperature (Tm) of DNA ▯ “temperature at which half the helical structure is lost” -Tm can be calculated from the change in free energy values of the component stacking interactions -“An all-or-none transition is a good model; at any given temperature, fully formed duplexes and separated single strands coexist” -“Only a small amount of partially-melted helices are ever present” -Helicases, not heat, are responsible for melting DNA double helices inside cells • Observing DNA melting - DNA absorbs light the greatest at a wavelength of 260 nm, the average wavelength for the four bases - Hypochromism: Increased base stacking (SS▯DS) ▯ decreased absorbance of ultraviolet light - Because double-helical DNA absorbs light less effectively than single-stranded DNA, one can observe the melting of DNA by monitoring the absorption of light, particularly at 260 nm • Annealing -The process by which complementary DNA strands recoil into a double helix when the temperature is lowered below Tm Note: Quotations indicate text obtained directly from textbook or lecture notes References Berg, Jeremy, John Tymoczko, and Lubert Stryer. Biochemistry. 7th ed. W.H. Freeman, 2012. 1- 246. Print 10


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