Protein Structure and Analysis lect 3
Recap of Amino Acids and Peptide Bonds
Amino acids form peptides, drawn from the N-terminus to the acid (C-terminus).
The amide bond (between amino acids) has a partially double-bonded nature due to nitrogen's electron delocalization, restricting rotation and resulting in a flat molecular structure.
Amides, unlike amines, are not readily protonated due to the adjacent carbonyl group, and they do not typically act as nucleophiles or bases.
Synthetic peptide synthesis involves activated carboxylic acid analogs with leaving groups, such as chlorides or anhydrides. DCC (dicyclohexylcarbodiimide) is used to activate the carboxylic acid.
Without protecting groups, random peptide chains can form. Controlled synthesis requires protecting groups to react amino acids sequentially, which is time-consuming but can be automated using solid-phase synthesis on microbeads.
Peptides have varying charges depending on side groups, leading to an isoelectric point (net neutral charge) that requires calculation.
Proteins: Long Peptide Sequences
Short peptides are important in biological signaling and secretions.
Forensic proteomics analyzes peptides and proteins in biological residues, offering a unique biological fingerprint.
DNA analysis is becoming less reliable due to trace contamination issues, making proteomics a valuable supplementary or standalone technique.
Proteomics is increasingly used in finger mark analysis, examining biological components like proteins and peptides.
Protein Structure Levels
Proteins have four levels of structure: primary, secondary, tertiary, and quaternary.
Primary Structure: The amino acid sequence from the N-terminus to the C-terminus. At this level, the full chemical structure is often simplified due to the size of the molecule.
Secondary Structure: Regular, repeating structural motifs formed by the amino acid backbone.
Tertiary Structure: The overall three-dimensional folding of the protein.
Quaternary Structure: The assembly of multiple protein subunits into a larger complex (e.g., enzymes with multiple protein components forming a molecular machine).
Secondary Structure and Amide Bonds
Secondary structure relies on hydrogen bonds between amide chains.
Hydrogen bonds are electrostatic interactions between partially positive hydrogen atoms and partially negative oxygen atoms.
Ramachandran Plots and Protein Conformations
Ramachandran plots analyze possible rotations along the protein chain.
The bonds can be rotated in different ways. The longer the chain, the number of possible confirmations increases exponentially.
Computer models are used to analyze protein conformations, considering steric hindrance between side groups.
Certain conformations are more stable due to lower energy. Analyzing every possible bond angle is computationally intensive.
Ramachandran plots confirm that alpha helices, beta sheets, and turns are the most common polypeptide chain conformations based on computer modeling.
Alpha Helix
Alpha helices are stabilized by hydrogen bonds along the chain between amide bonds.
Every seven amino acid residues, the helix returns to a similar point in space. Alpha helix is represented as helix shape biochemically.
Amino acids like methionine, alanine, and leucine promote alpha helix formation, though there's no clear common feature among them. Smaller groups are preferred.
Substituents in an alpha helix are kept as far apart as possible.
Beta Sheets
Beta sheets consist of long, relatively straight chains stacked next to each other with hydrogen bonding between different parts of the chain.
Chains can be in the same molecule (folding back) or different molecules and are represented by large arrows, indicating the direction of the amine to acid direction.
Beta sheets are two-dimensional and flat.
Bulky aromatic residues and branched amino acids generally promote beta sheet formation.
Turns and Other Structures
Beta turns allow beta sheets to fold back on themselves.
Elongated helices can form helices every 10 residues.
Triple helices and random coils (no defined conformation) can also occur.
Proteins often contain alpha helices, beta sheets, and random coils connecting structured elements.
Tertiary Structure and Side Group Interactions
Tertiary structure is determined by interactions between side groups.
Intermolecular forces: hydrogen bonding, hydrophobic effect, charged group interactions, and π-π stacking.
Covalent bonds: disulfide bridges between cysteine residues, forming a covalent sulfur-sulfur bond.
Disulfide Bonds
Cysteine can form disulfide bonds in a protein.
The disulfide bond is a relatively weak bond.
Oxidative addition: 2R-SH → R-S-S-R (disulfide bond).
Examples of Proteins and Their Structures
Lysozyme (antibacterial protein).
Chymotrypsin (digestive enzyme).
Membrane channel (transports substances across cell membranes).
Epidermal growth factor receptor (regulates growth and regeneration).
Coiled Coils
Coils that coil around each other can form molecular ropes, found in B silk. Spider silk is strong but expensive.
Researchers are exploring protein manipulation at the nanoscale for specialized applications.
Quaternary Structure: Multiple Polypeptide Chains
Multiple polypeptide chains come together via intermolecular forces.
Examples: insulin (two polypeptide chains), cytochrome c (five polypeptides).
Researchers study protein structures to understand their structure and function.
Protein Analysis Techniques
X-Ray Diffraction
X-ray diffraction is used for molecular identification.
Single crystal X-ray diffraction requires a crystal with a repeat unit cell.
Crystallization involves dissolving the protein in water and evaporating the water to allow crystal formation. This process can take weeks or months, varying factors like concentration, salts, ligands, precipitants, surfactants, temperature, and organic solvents.
The crystal is then frozen and analyzed using X-ray diffraction to determine the complete structure.
The process is highly complex and time-consuming, often resulting in non-crystalline samples or damaged crystals..
Gel Electrophoresis
Gel electrophoresis is used for protein and peptide analysis. It is automated and can be used for biological sample analysis.
A charged molecule in a electric field move according to its weight.
Polyacrylamide gel electrophoresis (PAGE) uses a hydrophilic gel with cathode and anode and an electrolyte solution. Sample is introduced, then an electric field is applied, and proteins are separated based on size. You can isolate these proteins for chemical analysis.
Sodium dodecyl sulfate (SDS) is used to give proteins an overall negative charge, ensuring separation is primarily based on size.
The unique patterns from various individuals will depend on how much they sweat and the composition of their metabolic processes.
NMR Spectroscopy
NMR is used to produce spectra. However, it is difficult to do on proteins.
2D NMR spectra of nitrogens and hydrogens and how they interact can be used to determine protein structure.
NMR analysis can take days to complete.
Cryo-Electron Microscopy
Cryo-electron microscopy visualizes protein structures by using electrons instead of light.
This is especially useful for proteins with regular shapes.
Protein Data Bank
The protein databank contains 200,000 protein structures.
It includes crystal, NMR data, and cryo-EM data.
AI Prediction: Artificial intelligence is revolutionizing structure prediction, generating protein structures from amino acid sequences potentially in days, which used to take decades.
Large linear learning models or AI increasingly can predict the exact protein structure that will result from the amino acid residues, turning decades of work into something that will take days.
Already 200,000,000 protein structure predictions have been made and they're being matched up to proteins you find in nature and generating a database for further protein structures that are highly likely.
Recap of Amino Acids and Peptide Bonds
Amino acids form peptides, drawn from the N-terminus to the acid (C-terminus). This directionality is crucial for understanding protein structure and function.
The amide bond (between amino acids) has a partially double-bonded nature due to nitrogen's electron delocalization, restricting rotation and resulting in a flat molecular structure. This rigidity influences protein folding.
Amides, unlike amines, are not readily protonated due to the adjacent carbonyl group, and they do not typically act as nucleophiles or bases. This property affects their chemical behavior in biological systems.
Synthetic peptide synthesis involves activated carboxylic acid analogs with leaving groups, such as chlorides or anhydrides. DCC (dicyclohexylcarbodiimide) is used to activate the carboxylic acid. Modern techniques also employ various coupling reagents like HATU or HBTU to improve efficiency and reduce racemization.
Without protecting groups, random peptide chains can form. Controlled synthesis requires protecting groups to react amino acids sequentially, which is time-consuming but can be automated using solid-phase synthesis on microbeads. Common protecting groups include Fmoc and Boc.
Peptides have varying charges depending on side groups, leading to an isoelectric point (net neutral charge) that requires calculation. The Henderson-Hasselbalch equation can be used to determine the charge at a given pH.
Proteins: Long Peptide Sequences
Short peptides are important in biological signaling and secretions. Examples include hormones and neurotransmitters.
Forensic proteomics analyzes peptides and proteins in biological residues, offering a unique biological fingerprint. This involves techniques like mass spectrometry to identify and quantify proteins.
DNA analysis is becoming less reliable due to trace contamination issues, making proteomics a valuable supplementary or standalone technique. Proteomics can identify individuals based on their unique protein profiles.
Proteomics is increasingly used in finger mark analysis, examining biological components like proteins and peptides. This can provide more detailed information than traditional fingerprinting methods.
Protein Structure Levels
Proteins have four levels of structure: primary, secondary, tertiary, and quaternary. Each level is essential for the protein's function.
Primary Structure: The amino acid sequence from the N-terminus to the C-terminus. At this level, the full chemical structure is often simplified due to the size of the molecule. This sequence dictates all higher-order structures.
Secondary Structure: Regular, repeating structural motifs formed by the amino acid backbone. Common motifs include alpha helices and beta sheets, stabilized by hydrogen bonds.
Tertiary Structure: The overall three-dimensional folding of the protein. This structure is stabilized by various interactions between side chains.
Quaternary Structure: The assembly of multiple protein subunits into a larger complex (e.g., enzymes with multiple protein components forming a molecular machine). Not all proteins have a quaternary structure.
Secondary Structure and Amide Bonds
Secondary structure relies on hydrogen bonds between amide chains. These bonds are crucial for the stability of alpha helices and beta sheets.
Hydrogen bonds are electrostatic interactions between partially positive hydrogen atoms and partially negative oxygen atoms. They are relatively weak but collectively strong.
Ramachandran Plots and Protein Conformations
Ramachandran plots analyze possible rotations along the protein chain, specifically the phi (\φ) and psi (\ψ) angles.
The bonds can be rotated in different ways. The longer the chain, the number of possible confirmations increases exponentially. Steric hindrance and other factors limit the accessible conformations.
Computer models are used to analyze protein conformations, considering steric hindrance between side groups. Molecular dynamics simulations can predict protein folding pathways.
Certain conformations are more stable due to lower energy. Analyzing every possible bond angle is computationally intensive. Energy minimization techniques are used to find stable conformations.
Ramachandran plots confirm that alpha helices, beta sheets, and turns are the most common polypeptide chain conformations based on computer modeling. These plots show the distribution of phi and psi angles for these structures.
Alpha Helix
Alpha helices are stabilized by hydrogen bonds along the chain between amide bonds. These bonds occur between the carbonyl oxygen of one amino acid and the amide hydrogen of another amino acid four residues down the chain.
Every seven amino acid residues, the helix returns to a similar point in space. Alpha helix is represented as helix shape biochemically. The pitch of the helix is about 5.4 Angstroms.
Amino acids like methionine, alanine, and leucine promote alpha helix formation, though there's no clear common feature among them. Smaller groups are preferred. Proline and glycine are often helix breakers.
Substituents in an alpha helix are kept as far apart as possible. This minimizes steric clashes and stabilizes the helix.
Beta Sheets
Beta sheets consist of long, relatively straight chains stacked next to each other with hydrogen bonding between different parts of the chain. These can be parallel or antiparallel.
Chains can be in the same molecule (folding back) or different molecules and are represented by large arrows, indicating the direction of the amine to acid direction. Antiparallel sheets are more stable due to better alignment of hydrogen bonds.
Beta sheets are two-dimensional and flat. The distance between adjacent amino acids in a beta sheet is approximately 3.5 Angstroms.
Bulky aromatic residues and branched amino acids generally promote beta sheet formation. Examples include valine, isoleucine, and tyrosine.
Turns and Other Structures
Beta turns allow beta sheets to fold back on themselves. These often involve proline or glycine residues.
Elongated helices can form helices every 10 residues. These are less common than alpha helices.
Triple helices and random coils (no defined conformation) can also occur. Collagen is a well-known example of a triple helix.
Proteins often contain alpha helices, beta sheets, and random coils connecting structured elements. These elements combine to form the overall tertiary structure.
Tertiary Structure and Side Group Interactions
Tertiary structure is determined by interactions between side groups. These interactions determine the protein's overall shape and function.
Intermolecular forces: hydrogen bonding, hydrophobic effect, charged group interactions, and π-π stacking. Van der Waals forces also play a role.
Covalent bonds: disulfide bridges between cysteine residues, forming a covalent sulfur-sulfur bond. These bonds are stronger than non-covalent interactions.
Disulfide Bonds
Cysteine can form disulfide bonds in a protein. This involves the oxidation of the thiol groups of two cysteine residues.
The disulfide bond is a relatively weak bond. However, it contributes significantly to protein stability.
Oxidative addition: 2R-SH → R-S-S-R (disulfide bond). This reaction typically occurs in the endoplasmic reticulum.
Examples of Proteins and Their Structures
Lysozyme (antibacterial protein). It has a globular structure with alpha helices and beta sheets.
Chymotrypsin (digestive enzyme). It has a beta-barrel structure and is involved in peptide bond hydrolysis.
Membrane channel (transports substances across cell membranes). These proteins often have transmembrane helices.
Epidermal growth factor receptor (regulates growth and regeneration). It has a complex structure with multiple domains.
Coiled Coils
Coils that coil around each other can form molecular ropes, found in B silk. Spider silk is strong but expensive. These structures are stabilized by hydrophobic interactions.
Researchers are exploring protein manipulation at the nanoscale for specialized applications. This includes creating new materials and devices.
Quaternary Structure: Multiple Polypeptide Chains
Multiple polypeptide chains come together via intermolecular forces. This forms a functional protein complex.
Examples: insulin (two polypeptide chains), cytochrome c (five polypeptides). Hemoglobin is another example with four subunits.
Researchers study protein structures to understand their structure and function. This knowledge is crucial for drug development and understanding disease mechanisms.
Protein Analysis Techniques
X-Ray Diffraction
X-ray diffraction is used for molecular identification. It provides high-resolution structures of proteins.
Single crystal X-ray diffraction requires a crystal with a repeat unit cell. The crystal must be of high quality for accurate data collection.
Crystallization involves dissolving the protein in water and evaporating the water to allow crystal formation. This process can take weeks or months, varying factors like concentration, salts, ligands, precipitants, surfactants, temperature, and organic solvents. Additives like PEG are often used.
The crystal is then frozen and analyzed using X-ray diffraction to determine the complete structure. The diffraction pattern is used to calculate the electron density map.
The process is highly complex and time-consuming, often resulting in non-crystalline samples or damaged crystals.. Data processing and structure determination require specialized software.
Gel Electrophoresis
Gel electrophoresis is used for protein and peptide analysis. It is automated and can be used for biological sample analysis. This technique separates proteins based on size and charge.
A charged molecule in a electric field move according to its weight. Smaller molecules move faster through the gel.
Polyacrylamide gel electrophoresis (PAGE) uses a hydrophilic gel with cathode and anode and an electrolyte solution. Sample is introduced, then an electric field is applied, and proteins are separated based on size. You can isolate these proteins for chemical analysis. The gel matrix is formed by polymerizing acrylamide and bis-acrylamide.
Sodium dodecyl sulfate (SDS) is used to give proteins an overall negative charge, ensuring separation is primarily based on size. SDS also denatures the proteins, unfolding them for better separation.
The unique patterns from various individuals will depend on how much they sweat and the composition of their metabolic processes. This makes it useful in forensic science.
NMR Spectroscopy
NMR is used to produce spectra. However, it is difficult to do on proteins. It provides information about the protein's dynamics and interactions.
2D NMR spectra of nitrogens and hydrogens and how they interact can be used to determine protein structure. Techniques like COSY, TOCSY, and NOESY are used to identify these interactions.
NMR analysis can take days to complete. The data analysis is complex and requires specialized expertise.
Cryo-Electron Microscopy
Cryo-electron microscopy visualizes protein structures by using electrons instead of light. The sample is flash-frozen to preserve its native state.
This is especially useful for proteins with regular shapes. It can also be used for large protein complexes and membrane proteins.
Protein Data Bank
The protein databank contains 200,000 protein structures. It is a valuable resource for researchers.
It includes crystal, NMR data, and cryo-EM data. The data is freely available to the public.
AI Prediction: Artificial intelligence is revolutionizing structure prediction, generating protein structures from amino acid sequences potentially in days, which used to take decades. Algorithms like AlphaFold have significantly improved the accuracy of protein structure prediction.
Large linear learning models or AI increasingly can predict the exact protein structure that will result from the amino acid residues, turning decades of work into something that will take days. This is transforming structural biology.
Already 200,