Biochemical Evolution

Key Points for CSIR NET from the Given Chapter:

1. Fossil Evidence of Early Life:

o Morphologically and biochemically similar organisms to modern bacteria existed 3.5

billion years ago, providing fossil evidence for the early evolution of life.

2. Hypothetical Evolutionary Path:

o The evolutionary path from the prebiotic world to modern organisms can be
hypothesized, though early events remain uncertain.

o Key evolutionary stages:

1. Generation of Key Molecules: Nonbiological processes produced nucleic

acids, proteins, carbohydrates, and lipids.

2. Prebiotic Chemistry to Replicating Systems: Transitioning from chemical

systems to biological systems that could replicate.

3. Energy Conversion and Biosynthesis: Evolution of mechanisms for energy

conversion (chemical sources, sunlight) and synthesis of key molecules.

4. Environmental Adaptation and Multicellularity: Organisms evolved

mechanisms to adjust biochemistry to changing environments, leading to
multicellular life forms.

3. Challenges in the Evolution of Life:

o The chapter introduces challenges of life's evolution, including how key biochemical
processes arose, which will be explored in detail later.

4. Synthesis of Organic Molecules:

o Before life, the synthesis of organic molecules from simpler molecules was necessary
for life to develop.

o Complex organic molecules needed for life (e.g., components of nucleic acids and
proteins) could be produced via prebiotic reactions.

5. Miller-Urey Experiment:

o In the 1950s, Stanley Miller and Harold Urey conducted experiments simulating early
Earth conditions (methane, ammonia, water, hydrogen, and lightning).

o They showed that amino acids (e.g., glycine, alanine) and other fundamental organic
compounds could be synthesized under these conditions.

o Amino acids like glutamic acid and leucine were also produced, and adenine could be
synthesized from hydrogen cyanide (HCN).
 o Sugars, including ribose (important for nucleic acids), could be formed from
formaldehyde in prebiotic conditions.

Likely Questions for CSIR NET:

 What are the stages in the hypothetical evolutionary path from the prebiotic world to
modern organisms?

 Explain the significance of the Miller-Urey experiment in understanding the origin of life.

 How could key organic molecules, such as nucleic acids and proteins, be produced under
prebiotic conditions?

 What role did early Earth's atmosphere play in the formation of complex biochemical
molecules?

Key Points for CSIR NET from the Given Section:

1. Uncertainties in Biomolecule Origins:

o While many building blocks of life (e.g., sugars, amino acids) were synthesized under
prebiotic conditions, uncertainties exist, particularly concerning ribose:

 Ribose is one of many sugars formed, but it is unstable under prebiotic

conditions.

 Ribose in RNA occurs in only one mirror-image form, posing challenges in its
prebiotic synthesis.

 It is speculated that early nucleic acids may have used a different backbone
before ribose was incorporated.

2. Principles of Evolution: Evolution requires three fundamental principles:

1. Reproduction/Replication:

 For evolution to occur, replicating molecules must exist. Without replication,

molecules degrade over time, preventing evolution.

 Biological polymers like RNA degrade via hydrolysis, but replicating systems
persist in populations.

2. Variation:

 Replication must introduce variations; perfect replication would halt

evolution. Variations provide the foundation for natural selection.

3. Competition and Selective Pressure:

 Replicating molecules or organisms compete for resources, leading to

natural selection.

 Variants better suited to survival and replication dominate, leading to

evolution of new, more efficient forms.

3. Molecular Evolution Demonstrated in Vitro (Spiegelman Experiment):

 o In 1967, Sol Spiegelman demonstrated molecular evolution using RNA from
bacteriophage Qβ.

o He mixed Qβ replicase with RNA molecules. Without selective pressure, the

population remained unchanged.

o When conditions such as replication time were limited, new species evolved, such as
RNA fragments replicating more efficiently (e.g., a smaller 550-base RNA replicating
15 times faster than the original).

o Spiegelman’s experiment highlights that selective pressures (e.g., limited precursors,

inhibitors) drive molecular evolution in vitro.

4. Self-Replication of RNA:

o The molecular structure of nucleic acids, particularly RNA, suggests they could self-
replicate.

o Some studies show single-stranded nucleic acids can serve as templates for
complementary strand synthesis spontaneously (without external machinery).

o However, no conditions have yet been found where RNA can fully self-replicate from
simple starting materials.

Likely Questions for CSIR NET:

 What are the uncertainties regarding the prebiotic synthesis of ribose and nucleic acids?

 Discuss the fundamental principles necessary for evolution at the molecular level.

 What are the implications of the Spiegelman experiment in understanding molecular

evolution?

 How does RNA serve as a template for replication, and what challenges remain in finding
conditions for independent RNA self-replication?

Key Points for CSIR NET from the Given Section:

1. RNA Molecules as Catalysts (Ribozymes):

o RNA molecules, in addition to storing genetic information, can act as catalysts, a

discovery made by Tom Cech and Sidney Altman in the 1980s.

o Catalytic RNA molecules are called ribozymes, and they can promote specific
biochemical reactions.

o Example: The hammerhead ribozyme found in plant viruses promotes RNA cleavage,
highlighting the potential catalytic role of RNA in early evolution.

o This supports the hypothesis of an early RNA world, where RNA played a central role
in heredity, storage of information, and catalysis.

2. Amino Acids and Their Role in Biosynthesis and Catalysis:

 o As RNA replication increased, the prebiotic building blocks of RNA became limited,
which may have driven the evolution of mechanisms to synthesize them, including
the use of amino acids.

o Amino acids were likely involved in the early synthesis of RNA bases (e.g., purines
and pyrimidines).

o Polypeptides (short chains of amino acids) may have contributed to ribozymes by

adding chemical versatility, enhancing their catalytic functions.

o Unlike nucleic acids, polypeptides can fold into three-dimensional structures due to
the variety in amino acid side chains, allowing for more complex biochemical
interactions.

3. RNA-Directed Polypeptide Synthesis (Linking RNA and Protein Worlds):

o Initially, polypeptides were produced indirectly by RNA molecules, which favored the
survival of certain RNA sequences by promoting the synthesis of specific
polypeptides.

o A critical evolutionary step occurred when an apparatus evolved allowing RNA to

directly dictate the sequence of polypeptides. This gave rise to the genetic code,
where three bases (a codon) in RNA correspond to a specific amino acid.

o The ribosome, a complex composed largely of RNA (ribosomal RNA, or rRNA), is

responsible for catalyzing polypeptide synthesis and acts as a highly evolved
ribozyme.

o tRNA molecules serve as adapters that bring specific amino acids to the ribosome,
where peptide bonds are formed, creating the growing polypeptide chain.

Likely Questions for CSIR NET:

 What are ribozymes, and how do they contribute to the hypothesis of an RNA world?

 Discuss the role of amino acids in early biosynthetic processes and how they contributed to
the evolution of complex biochemical pathways.

 Explain how the transition from RNA-directed polypeptide synthesis to the modern genetic
code occurred.

 How does the ribosome function as a ribozyme in modern organisms, and what does this
suggest about its evolutionary history?

Key Points for CSIR NET from the Given Section:

1. The Genetic Code and Evolution:

o A gene is a sequence of DNA bases that encodes a functional protein.

o The genetic code is universal across all organisms, with few exceptions, suggesting it
was fixed early in evolution.

o Mutations (changes in the genetic sequence) are a primary source of variation in

evolution. These can be point mutations (single base change), insertions, or
deletions.
 o Gene duplication is another mechanism for evolution, where duplicated genes can
mutate and evolve new functions, allowing for biochemical and functional diversity.

2. Gene Duplication and Specialization:

o Gene duplication is a significant evolutionary process, where duplicated sections of

genetic material allow for the development of new biochemical functions without
starting from scratch.

o Duplication and divergence of genes have led to families of related enzymes and
proteins in modern organisms.

3. Transfer RNAs (tRNAs) and Gene Duplication:

o tRNA molecules, essential for protein synthesis, are similar in structure but differ
subtly to match their specific amino acid.

o The family of tRNAs likely evolved through gene duplication followed by

specialization, resulting in tRNAs for the 20 different amino acids used in protein
synthesis.

4. DNA as a Stable Genetic Storage Molecule:

o RNA was likely the original genetic material, but DNA (deoxyribonucleic acid)
replaced RNA because it is chemically more stable.

o DNA lacks the 2'-hydroxyl group found in RNA, which makes RNA more prone to
hydrolytic cleavage.

o DNA’s thymine (a methylated version of uracil) helps protect the integrity of the
genetic sequence, aiding in DNA repair mechanisms.

5. RNA’s Continuing Role in Modern Biology:

o Despite DNA taking over the role of storing genetic information, RNA continues to
play vital roles in modern organisms. It acts as:

 A template for protein synthesis (mRNA),

 The adapter in translation (tRNA),

 A catalytic component of the ribosome (rRNA).

6. Evolutionary Transition from RNA to DNA:

o In modern organisms, DNA is synthesized from RNA building blocks through enzymes
called ribonucleotide reductases.

o The evolutionary shift from RNA to DNA increased genetic stability, crucial for the
accurate transmission of genetic information across generations.

Likely Questions for CSIR NET:

 Explain how the genetic code demonstrates the universality of evolution.

 Describe the process and significance of gene duplication in evolution.

  How do tRNAs illustrate evolution through gene duplication and specialization?

 Discuss the advantages of DNA over RNA as the genetic material.

 What roles does RNA still play in modern organisms, despite DNA taking over genetic
information storage?

Key Concepts:

1. ATP as a Common Energy Currency: ATP is the main molecule that stores and transfers
energy in cells. It contains three phosphates, and when its bonds are broken, it releases
energy to drive other processes.

2. ATP Generation: ATP is produced by breaking down molecules like amino acids and sugars
(e.g., glycine, glucose). Early life forms likely used amino acids for ATP production, but later
evolved to utilize glucose in processes like glycolysis.

3. Cell Membrane Formation: Cells are enclosed by membranes composed of lipids. Lipid
bilayers are formed from amphipathic molecules, which have both hydrophilic and
hydrophobic parts. These bilayers allow for compartmentalization of cellular contents,
essential for maintaining cell integrity and function.

4. Osmotic Challenges and Ion Pumps: Early cells faced osmotic pressures due to their semi-
permeable membranes. Ion pumps evolved to counteract this, using energy (usually from
ATP) to maintain ion gradients that balance water flow and prevent cells from bursting.

5. Proton Gradients and ATP Synthesis: Proton gradients across membranes can drive ATP
synthesis. This process, facilitated by ATP synthase enzymes, allows cells to generate ATP by
harnessing energy from the movement of protons.

6. Photosynthesis and Oxygen: Photosynthesis evolved to use sunlight to drive electron

transfer across membranes, creating proton gradients for ATP synthesis. In some
photosynthetic organisms, water is used as an electron donor, producing oxygen as a by-
product. Oxygen later played a crucial role in the evolution of aerobic organisms, which use
oxygen to efficiently produce ATP from glucose.

7. Response to Environmental Changes

8. Cells adapt to their changing environments through mechanisms like altering enzyme activity,
synthesizing new enzymes, and changing membrane transport processes. Early detection
occurred inside cells via chemicals that passed through the membrane to bind with proteins.
An example is E. coli, which uses arabinose as an energy source by synthesizing enzymes
when arabinose binds to the AraC protein inside the cell. Over time, surface-based
mechanisms evolved, allowing cells to detect external signals via receptor proteins, initiating
internal changes, often involving "second messengers" like cyclic AMP (cAMP).
9. Intracellular and Cellular Movement
10. Movement is critical for adapting to environmental changes. Bacteria like E. coli use flagella
powered by proton gradients to move. Eukaryotic cells use microfilaments (actin) and
microtubules (tubulin) to enable movement. These structures serve as tracks for molecular
motors powered by ATP hydrolysis, facilitating intracellular transport and movement.
11. Formation of Colonies with Specialized Functions
12. Some cells form multicellular colonies, interacting through signaling mechanisms.
Dictyostelium is an example that can switch from single-cell existence to a multicellular
colony under starvation. This transition is driven by the release of cAMP, which signals other
cells to aggregate and form structures like fruiting bodies, helping spread cells to nutrient-rich
areas. This process of aggregation and differentiation foreshadows multicellular life.
 13. 2.4.3 The Development of Multicellular Organisms Requires Orchestrated Differentiation of
Cells
14. Multicellular organisms emerged around 600 million years ago. These organisms consist of
many distinct cell types, each specialized for different functions, even though the DNA
sequence within every cell is identical. This specialization is a result of differential gene
expression. Development begins from a single cell, which undergoes a series of regulated
processes, including gene expression, cell division, and movement, to form a complex
organism. The interactions and signaling between cells during embryogenesis play a crucial
role in guiding the cells to develop into specific tissues and organs.
15. The nematode Caenorhabditis elegans is a model organism extensively studied to understand
developmental biology. It has 959 cells, and its entire cell lineage, from the fertilized egg to
the adult, has been mapped. The development of C. elegans involves not only cell division
but also programmed cell death (apoptosis), indicating that the death of specific cells is
essential for proper development. Many of the genes and pathways involved in the
development of this simple organism are evolutionarily conserved in more complex
organisms, including humans.
16. 2.4.4 The Unity of Biochemistry Allows Human Biology to Be Effectively Probed Through
Studies of Other Organisms
17. All living organisms share a common evolutionary origin, which is reflected in the unity of
their biochemical processes. Complex organisms like humans evolved through variations and
adaptations of fundamental biochemical pathways that were established early in life’s history.
The complexity seen in higher organisms often arises from gene duplication events, where
duplicated genes can specialize for new functions.
18. A prime example is the protein kinases, a class of enzymes involved in signal transduction
and regulation of cell growth and differentiation. The human genome encodes around 500
protein kinases, while even simple organisms like yeast have over 100. Despite their
diversity, all these kinases evolved from a common ancestral enzyme. Therefore, by studying
the properties of a single kinase, scientists can infer general principles applicable across this
family of proteins.
19. Many biological processes were first deciphered in simple model organisms. For example, the
early stages of embryonic development were studied in fruit flies, while yeast was
instrumental in understanding DNA replication and cell cycle control. Researchers can
manipulate genes in mice to explore gene function in mammals, leveraging the evolutionary
relationships to investigate complex biological functions and diseases in humans.

Protein Structure and Function

1. Functions of Proteins

 Functions:

o Catalysts: Proteins act as enzymes that accelerate biochemical reactions.

o Transport and Storage: Proteins transport and store molecules, such as oxygen.

o Support and Protection: They provide mechanical support (e.g., cytoskeleton) and
immune protection.
 o Movement: Proteins are involved in movement generation.

o Signal Transmission: They transmit nerve impulses and participate in cellular

signaling.

o Growth Control: Proteins regulate growth and differentiation.

 Possible Questions:

o What are the key functions of proteins in biological systems?

o How do enzymes accelerate biochemical reactions?

o Discuss the role of proteins in cellular signaling and communication.

2. Key Properties of Proteins

 Properties:

o Structure: Proteins are linear polymers of amino acids, with their function
dependent on their three-dimensional structure. The sequence of amino acids
dictates the folding and overall shape of the protein.

o Functional Groups: Proteins contain various functional groups that contribute to

their chemical reactivity and functional diversity.

o Interactions: Proteins can form complex assemblies, enhancing functionality through

synergistic interactions.

o Rigidity vs. Flexibility: Proteins can be rigid (structural roles) or flexible (functional
roles), affecting their interactions and activities.

 Possible Questions:

o How does the sequence of amino acids influence the structure and function of
proteins?

o What are the differences between rigid and flexible proteins in terms of their
biological roles?

o Explain how protein interactions lead to complex assemblies and enhanced

functionality.

3. Amino Acids as Building Blocks

 Structure of Amino Acids: Each amino acid contains a central carbon atom bonded to an
amino group, a carboxylic acid group, a hydrogen atom, and a distinctive R group (side
chain).

 Chirality: Most amino acids are chiral, with L isomers being the only ones used in proteins.

 Zwitterions: Amino acids exist as zwitterions at neutral pH, influencing their chemical
behavior.

 Possible Questions:

o Describe the structure of an α-amino acid and its significance in protein formation.
 o Why are only L-isomers of amino acids used in protein synthesis?

o What is the significance of the zwitterionic form of amino acids in physiological pH?

4. Diversity of Amino Acids

 Diversity:

o Twenty Standard Amino Acids: All proteins are constructed from 20 amino acids,
which differ in size, shape, charge, and reactivity.

o Hydrophobic and Hydrophilic Properties: Amino acids can be hydrophobic (e.g.,

valine, leucine) or hydrophilic (e.g., serine, threonine), impacting protein structure
and function.

o Reactive Groups: Some amino acids contain functional groups (e.g., thiols in
cysteine) that can form disulfide bonds, stabilizing protein structure.

 Possible Questions:

o How do the properties of amino acid side chains influence protein folding and
stability?

o Discuss the significance of hydrophobic and hydrophilic interactions in protein

structure.

o What role do disulfide bonds play in protein stabilization?

5. Ionizable Side Chains

 Ionizable Amino Acids: Seven amino acids (e.g., lysine, arginine, histidine, aspartic acid, and
glutamic acid) have ionizable side chains that can donate or accept protons.

 pKa Values: The ionization states of these amino acids depend on pH, influencing protein
behavior in different environments.

 Possible Questions:

o Which amino acids have ionizable side chains, and what are their roles in enzyme
activity?

o How do pKa values influence the charge state of amino acids at physiological pH?

o Explain the significance of histidine in enzyme active sites.

6. Abbreviations and Symbols

 Abbreviations: Each amino acid has a three-letter abbreviation and a one-letter symbol,
which are essential for communication in biochemistry.

 Possible Questions:

o What are the standard abbreviations used for amino acids in biochemical literature?

o Why are one-letter and three-letter codes important in protein structure analysis?

7. Evolutionary Perspective
  Evolution: The specific set of 20 amino acids likely emerged due to their diverse properties,
availability from prebiotic chemistry, and reduced reactivity compared to potential
alternatives.

 Possible Questions:

o Discuss the evolutionary significance of the 20 amino acids used in protein synthesis.

o What factors might have influenced the selection of specific amino acids during early
evolutionary processes?

3.2 PRIMARY STRUCTURE: AMINO ACIDS LINKED BY PEPTIDE BONDS

 Peptide Bond Formation:

o Proteins are linear polymers formed by linking the α-carboxyl group of one amino
acid to the α-amino group of another, resulting in a peptide bond (amide bond). This
process results in the formation of a dipeptide and involves the loss of a water
molecule.

o The reaction favors hydrolysis, requiring energy input for peptide bond formation,
yet peptide bonds are kinetically stable with a lifetime of up to 1000 years in
aqueous solutions without a catalyst.

 Polarity and Naming:

o A polypeptide chain exhibits polarity, with an α-amino group at one end (N-terminal)
and an α-carboxyl group at the other end (C-terminal). The sequence of amino acids
is conventionally written from N-terminal to C-terminal.

o Example: In the pentapeptide Tyr-Gly-Gly-Phe-Leu (YGGFL), tyrosine is the N-terminal

residue and leucine is the C-terminal residue.

 Polypeptide Structure:

o A polypeptide consists of a main chain (backbone) and variable side chains. The
backbone has hydrogen-bonding potential, with carbonyl groups acting as hydrogen-
bond acceptors and NH groups (except proline) as donors.

o Most polypeptides contain 50 to 2000 amino acid residues, and proteins typically
have molecular weights between 5,500 and 220,000 daltons (kd).

 Cross-linking:

o Some proteins have cross-linked structures, primarily through disulfide bonds

formed by the oxidation of cysteine residues, resulting in cystine. Extracellular
proteins often contain several disulfide bonds, whereas intracellular proteins
typically do not.

 Possible Questions:

o What is the significance of peptide bonds in protein structure?

o Describe the polarity of a polypeptide chain and how it is represented.

o Explain the role of disulfide bonds in protein stability.

3.2.1 Proteins Have Unique Amino Acid Sequences Specified by Genes

 Historical Context:

o Frederick Sanger’s 1953 study on insulin demonstrated that proteins have a precisely
defined amino acid sequence, marking a significant advancement in biochemistry.

 Genetic Determination:

o The amino acid sequence of a protein (its primary structure) is genetically

determined by the nucleotide sequence in DNA, which is transcribed to RNA,
ultimately specifying the amino acid sequence. Each of the 20 amino acids is
encoded by specific triplets of nucleotides.

 Importance of Amino Acid Sequences:

o Understanding a protein’s amino acid sequence is crucial for elucidating its

mechanism of action, generating proteins with novel properties, and determining its
three-dimensional structure.

o Alterations in amino acid sequences can lead to diseases such as sickle-cell anemia
and cystic fibrosis. The amino acid sequence also provides insights into the
evolutionary history of proteins.

 Possible Questions:

o How did Frederick Sanger’s work contribute to our understanding of protein

structure?

o What is the link between DNA sequence and protein amino acid sequence?

o Discuss the implications of amino acid sequence changes in molecular pathology.

3.2.2 Polypeptide Chains Are Flexible Yet Conformationally Restricted

 Peptide Bond Geometry:

o The peptide bond is planar due to its double-bond character, preventing rotation
around it and constraining the conformation of the peptide backbone.

o The bond length between carbonyl and NH groups is shorter than typical single
bonds, and the peptide bond is uncharged, allowing tight packing of polypeptide
chains into globular structures.

 Trans and Cis Configurations:

o Most peptide bonds are in the trans configuration, minimizing steric clashes between
side chains. The rare cis configurations typically occur at proline residues due to its
unique bonding.

 Freedom of Rotation:

o The bonds between the amino group and the α-carbon and the α-carbon and
carbonyl group are single bonds, allowing rotation. This freedom permits various
orientations in polypeptide folding.
  Dihedral Angles:

o The rotation around these bonds can be defined by dihedral angles: phi (ϕ) for the
bond between nitrogen and the α-carbon, and psi (ψ) for the bond between the α-
carbon and carbonyl carbon.

o The Ramachandran diagram illustrates the allowed (ϕ, ψ) combinations, revealing

steric clashes that restrict many conformations.

 Folding Dynamics:

o Protein folding is thermodynamically favorable despite the entropy of an unfolded

random coil. The rigidity of peptide units and the restricted (ϕ, ψ) angles reduce
accessible structures, enabling protein folding.

 Possible Questions:

o Describe the geometric properties of peptide bonds and their significance in protein
structure.

o What are the allowed dihedral angles in polypeptide chains, and how do they
influence protein folding?

o Explain the role of steric exclusion in determining the conformation of polypeptide

chains.

3.3 SECONDARY STRUCTURE: POLYPEPTIDE CHAINS CAN FOLD INTO REGULAR STRUCTURES SUCH
AS THE ALPHA HELIX, THE BETA SHEET, AND TURNS AND LOOPS

3.3.1 The Alpha Helix Is a Coiled Structure Stabilized by Intrachain Hydrogen Bonds

In 1951, Linus Pauling and Robert Corey proposed that a polypeptide chain could fold into periodic
structures, notably the alpha helix (α-helix) and beta pleated sheet (β-pleated sheet). The α-helix is a
coiled structure where the backbone forms the inner part and the side chains extend outward. This
structure is stabilized by hydrogen bonds between the NH and CO groups of the main chain.
Specifically, each CO group forms a hydrogen bond with the NH group of the amino acid four
residues ahead in the sequence.

 Structural Characteristics:

o Each residue contributes to a rise of 1.5 Å along the helix axis.

o The rotation is 100 degrees, resulting in 3.6 amino acid residues per turn.

o The total pitch of the α-helix is 5.4 Å.

The α-helix can be either right-handed (clockwise) or left-handed (counterclockwise), but right-
handed helices are energetically more favorable due to less steric clash between side chains and the
backbone.

The α-helical content in proteins varies widely, from none to nearly 100%. For instance, ferritin
contains about 75% α-helices. In larger proteins, two or more α-helices can entwine to form stable
structures, as seen in myosin, tropomyosin, and keratin.

3.3.2 Beta Sheets Are Stabilized by Hydrogen Bonding Between Polypeptide Strands
Alongside the α-helix, Pauling and Corey identified the β-pleated sheet (β-sheet). Unlike the coiled α-
helix, the β-strand in a β-sheet is almost fully extended.

 Structural Characteristics:

o The distance between adjacent amino acids along a β-strand is approximately 3.5 Å.

o Side chains of adjacent amino acids point in opposite directions.

A β-sheet can form through hydrogen bonding between two or more strands, which can run in either
parallel or antiparallel directions:

 Antiparallel β-sheet: The NH and CO groups of each amino acid are hydrogen bonded to the
CO and NH groups of a partner on an adjacent strand.

 Parallel β-sheet: The NH group is hydrogen bonded to the CO group of one amino acid on
the adjacent strand, while the CO group is hydrogen bonded to the NH group of an amino
acid two residues further along.

Typically, 4 to 10 strands can combine to form β-sheets, which are depicted by broad arrows in
schematic diagrams pointing toward the carboxyl-terminal end.

3.3.3 Polypeptide Chains Can Change Direction by Making Reverse Turns and Loops

Proteins often require changes in the direction of their polypeptide chains to maintain compact,
globular shapes. These reversals are commonly achieved through reverse turns (also known as turns
or hairpin bends).

 Reverse Turns: Often stabilize abrupt changes in direction through hydrogen bonding
between the CO group of residue i and the NH group of residue i + 3.

 Loops: More complex structures that do not have regular periodic structures but are often
rigid and well-defined. Loops typically lie on protein surfaces and participate in molecular
interactions.

 3.4 Tertiary Structure: Water-Soluble Proteins Fold into Compact

Structures with Nonpolar Cores
 Proteins, when fully folded, exhibit complex three-dimensional structures that are
crucial for their functions. Myoglobin, an oxygen-carrying protein found in muscle,
serves as a prime example. Comprising a single polypeptide chain of 153 amino acids,
myoglobin's oxygen-binding capacity is attributed to its heme group, which contains
protoporphyrin IX and a central iron atom.
 Structure of Myoglobin
 Myoglobin is a compact molecule with dimensions of approximately 45 x 35 x 25 Å.
About 70% of its structure is comprised of eight α-helices, while the remaining
segments form loops and turns. The tertiary structure, which describes the overall
folding of the polypeptide chain, is characterized by a distinct arrangement of side
chains: the interior primarily consists of nonpolar residues (e.g., leucine, valine,
methionine, phenylalanine), while polar and charged residues are mainly located on
the surface. Histidine residues, however, are found in the interior, where they are
essential for binding iron and oxygen.
 This distribution of polar and nonpolar residues highlights a fundamental aspect of
protein architecture. In an aqueous environment, proteins tend to fold such that
hydrophobic (nonpolar) side chains are sequestered from water, leading to a more
 thermodynamically stable configuration. This process involves burying hydrophobic
residues while presenting polar and charged groups on the protein surface.
 3.5 Quaternary Structure: Polypeptide Chains Can Assemble into
Multisubunit Structures
 Proteins with multiple polypeptide chains exhibit a fourth level of structure known as
quaternary structure. Each individual polypeptide in such proteins is termed a subunit.
The simplest quaternary structure is a dimer, which consists of two identical subunits.
More complex forms can include varying types and numbers of subunits. For
instance, human hemoglobin is a tetramer composed of two α and two β subunits,
allowing it to efficiently transport oxygen throughout the body.
 3.6 The Amino Acid Sequence of a Protein Determines Its Three-
Dimensional Structure
 Anfinsen's Experiment
 The relationship between a protein's amino acid sequence and its three-dimensional
conformation was famously demonstrated by Christian Anfinsen with ribonuclease,
an enzyme made up of 124 amino acids. Anfinsen's experiments involved denaturing
the enzyme and subsequently allowing it to refold. Remarkably, the enzyme regained
its activity upon removal of denaturing agents, suggesting that the sequence of amino
acids contains all necessary information for proper folding.
 Anfinsen observed that when ribonuclease was reduced and then reoxidized in the
presence of denaturants, it formed incorrect disulfide bonds and retained only 1% of
its enzymatic activity. However, under favorable conditions, such as the presence of
trace amounts of a reducing agent, scrambled ribonuclease could convert to its native,
enzymatically active form.
 Amino Acid Preferences and Secondary Structure
 The propensity of specific amino acids to form α-helices, β-sheets, and turns provides
insights into protein folding. For example, residues like alanine and leucine are often
found in α-helices, while valine and isoleucine favor β-strands. Glycine and proline
are commonly found in turns due to their unique structural characteristics. Predictions
of secondary structures based on these preferences achieve around 60-70% accuracy.
 3.6.3 Protein Folding: Cooperative Process and Kinetics
 Protein folding is not random; it is a cooperative process where the stability of one
part of the protein influences others. As a result, proteins often undergo an "all or
none" transition during folding, meaning that they exist in either a fully folded or fully
unfolded state rather than in a mixed state.
 Levinthal's paradox illustrates the improbability of proteins folding by randomly
sampling all possible conformations due to the astronomical number of potential
structures. Instead, proteins fold through a series of partially correct intermediates,
allowing them to efficiently arrive at their native conformations. This concept
emphasizes the significance of retaining stable intermediates during the folding
process.

3.6.4 Prediction of Three-Dimensional Structure from Sequence

 Protein Structure Determination: The three-dimensional structure of a protein is

determined entirely by its amino acid sequence, but predicting this structure is a significant
challenge.

 Secondary Structure: Local sequences can predict only 60-70% of the secondary structure,
with long-range interactions necessary for determining the full tertiary structure.
  Prediction Approaches:

o Ab Initio Prediction: This method predicts protein folding without reference to

known structures, using computer calculations to minimize free energy or simulate
folding. Limitations include the vast number of possible conformations and the
marginal stability of proteins.

o Knowledge-Based Methods: These methods leverage existing knowledge of protein

structures. They assess an unknown sequence for compatibility with known
structures, allowing significant matches to serve as initial models for predicting
three-dimensional conformation.

3.6.5 Protein Modification and Cleavage Confer New Capabilities

 Covalent Modifications: Many proteins undergo covalent modifications that enhance their
functionality:

o Acetylation: Acetyl groups added to amino termini increase protein stability against
degradation.

o Hydroxylation: Hydroxyl groups added to proline residues stabilize collagen fibers,

with vitamin C deficiency leading to weakened collagen and conditions like scurvy.

o Carboxylation: Insufficient carboxylation of glutamate in prothrombin due to vitamin

K deficiency can cause bleeding disorders.

o Glycosylation: Carbohydrate units added to asparagine residues increase

hydrophilicity and interaction capability of proteins.

o Fatty Acid Addition: Fatty acids added to amino groups or cysteine increase
hydrophobicity.

 Phosphorylation: This modification alters enzyme activity, with serine and threonine
commonly phosphorylated. Phosphorylation acts as a reversible switch in cellular regulation.

 Chemical Rearrangements: Certain proteins, like the fluorescent green protein from jellyfish,
derive their properties from spontaneous rearrangements of side chains.

 Cleavage and Trimming: Proteins are often cleaved post-synthesis to become active.
Digestive enzymes are initially inactive precursors, and peptide-bond cleavage is essential for
processes like blood clotting and the activation of hormones.