Lecture 4_BIOC212_Maria Vera Ugalde_2025 - Copy

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Title

• Protein Folding in the Cell - 2

• Course: BIOC212 Winter 2025

• Instructor: Maria Vera Ugalde

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Summary from Lecture 3

• Amino acids (AA) bind through peptide bonds; sequence determines protein folding into functional 3-D structures.

• Both AA peptide bonds and side chains engage in non-covalent interactions to form secondary, tertiary, and quaternary protein structures.

• The side chain (R) influences protein charge, polarity, and hydrophobicity, affecting interactions.

• Similar AAs provide homology among protein family members or conserved proteins through evolution:

  • Types of AAs:

    • Hydrophobic

    • Polar

    • Basic

    • Acidic

• Proteins are polymers made of 20 different amino acids, categorized by functional group of side chains.

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Sequence Similarity

• Sequences of polypeptides can be compared to align identical and similar amino acids.

• Sequence similarity (homology) indicates evolutionary conservation.

• Homology suggests common structure or function.

• Polypeptides without sequence similarity are divergent.

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Example of Sequence Similarity

• Example sequences from Homo sapiens and Apis mellifera:

  • Alignment reveals identity, strong similarity, and weak similarity.

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Amino Acid Side Chains

• Uncharged Polar Side Chains:

  • Asparagine (Asn, N), Glutamine (Gln, Q)

• Nonpolar Side Chains:

  • Valine (Val, V), Alanine (Ala, A), Leucine (Leu, L), Glycine (Gly, G), Cysteine (Cys, C), Isoleucine (Ile, I)

• Basic Side Chains:

  • Lysine (Lys, K), Arginine (Arg, R), Histidine (His, H)

• Acidic Side Chains:

  • Aspartic acid (Asp, D), Glutamic acid (Glu, E), Methionine (Met, M)

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Protein Families

• A family consists of proteins or domains with homologous sequences and structures, often with related functions.

• An organism may have multiple proteins from the same family, found across different organisms.

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Topics for Discussion

• Post translational modifications of AAs.

• Protein folding processes.

• Protein misfolding.

• Protein quality control mechanisms.

• Assisted folding by chaperones.

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Post-Translational Modification (PTM)

• Proteins can undergo chemical modification post-translation, contributing to diversity and regulating function.

• Types of modifications include:

  • Cleavage into smaller proteins by peptidases.

  • Covalent modifications of N-terminus or side chains.

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Functions of Side Chain Modifications

• Can alter protein surface or conformation, create/block binding sites, and are often regulated and reversible.

• Modifications are swift, acting as functional switches.

• Main types: Phosphorylation, Methylation, Acetylation, Glycosylation, Sumoylation, Ubiquitination.

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Phosphorylation

• Major regulatory mechanism involving phosphorylation of hydroxyl groups (S, T, Y).

• Kinases transfer phosphates from ATP and are specific to side chains and surrounding sequences.

• Phosphatases remove phosphate groups.

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Phosphorylation Enzymes

• Kinase Families:

  • Ser/Thr kinases, Tyr kinases, dual specificity kinases.

• Phosphatase Families:

  • Ser/Thr phosphatases, protein Tyr phosphatases, dual specificity phosphatases.

• Study of phosphorylation's role in protein function; phosphomimic and de-phosphorylated strategies.

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Phosphopeptide Binding

• Specialized domains bind phosphorylated serine, threonine, or tyrosine.

• Phosphorylation is vital for binding; surrounding polypeptide sequences also contribute.

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Acetylation of Lysine

• Acetylation changes polarity and involves lysine acetyltransferases (KATs) and deacetylases (KDACs).

• Effects include increased size and charge, altering function such as in histone acetylation.

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Methylation of Lysine & Arginine

• Methylation adds 1-2 methyl groups, creating methylarginines.

• Lysine can be mono-, di-, or trimethylated; involves specific methyltransferases and demethylases.

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PTM and Binding

• Acetylation and methylation provide new binding sites; specific domains must bind modified residues and surrounding sequences.

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Protein Folding and Structure

• Folded protein structure derives from hydrophobic interactions internally, with polar side chains on the exterior.

• The native state represents the fully folded conformation of a protein, where modifications may occur post-folding.

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Importance of Interactions for Folding

• Hydrogen bonds stabilize secondary structures and involve peptide bonds.

• Hydrophobic interactions dictate tertiary structures and involve side chains.

• Various bonds (hydrogen, ionic, disulfide) contribute to protein stability.

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Relationship Between AA Sequence and 3D Structure

• Native structure determined by primary AA sequence; represents a state of minimal energy and is thermodynamically favored.

• Although folding can be spontaneous, biological processes assist in folding.

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The Folding Process

• Folding is complex and transitions through intermediates with increasing structure toward the native state.

• Unfolded domains show extended conformations lacking secondary or tertiary structure.

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Native State of Proteins

• The native state is the most stable conformation stabilized by hydrophobic contacts and sometimes requires a ligand partner.

• Equilibrium can exist between native and near-native states.

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Folding Intermediates

• Intermediates have some secondary structure but incomplete tertiary structure, exposing hydrophobic regions.

• Increased flexibility leads to aggregation risks and insolubility due to hydrophobic contact preferences.

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Protein Misfolding Issues

• Causes of misfolding include incomplete folding, unavailable ligands, or genetic mutations.

• Misfolded proteins may lead to diseases and are exacerbated by environmental factors, aging, and decreased protein quality control efficiency.

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Genetic Mutations

• Mutations alter polypeptide sequences (substitution, insertion, deletion) and affect protein folding or function.

• Some mutations are pathogenic, while others may not show significant effects.

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Protein Production Rates

• Nucleus: ~1000 transcripts/second.

• Total mRNAs and proteins reach 10^5-10^6 and 10^10, respectively.

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Protein Quality Control and Homeostasis

• Chaperones are central to maintaining protein quality, assisted by proteasomes and autophagy.

• Proteostasis ensures correct protein concentration, conformation, and location, vital for proteome stability.

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Chaperones and Their Role

• Molecular chaperones aid in folding and prevent aggregation without integrating into the native state; often recognize exposed hydrophobic regions.

• Heat shock proteins (HSP) are key chaperones expressed during stress conditions.

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Stress Response Mechanisms

• Cells respond to stress via increased expression of chaperones, forming the Heat Shock Response (HSR) and Unfolded Protein Response (UPR).

• HSR protects against cell death; UPR manages ER protein stress.

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Activation of Chaperones

• Inducible chaperones are activated by heat and stress; constitutive chaperones assist general protein folding under non-stress conditions.

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Unfolded Protein Activation

• Unfolded cytosolic proteins activate heat shock responses and transcription of HSPs, adjusting protein synthesis based on stress severity.

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HSF1 and Heat Shock Response

• HSF1 mediates heat shock response and is regulated through interactions with Hsp90, influencing its activation and down-regulation mechanisms.

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Regulation of HSF

• Active HSF1 trimerizes for transcription activation after heat shock, whereas monomers bind Hsp90 to maintain its inactive state.

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Phosphate Binding Amino Acids

• Identifying amino acids that bind phosphate groups, specifically phospho-tyrosine.

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Thought Questions

1. Identify amino acids involved in binding to phospho-serine.

2. Compare recognition between phospho-serine and phospho-tyrosine.

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