4.1 Levels of protein structure and folding mechanisms

Proteins are the workhorses of life, and their structure is key to their function. From simple chains of amino acids to complex 3D shapes, proteins fold into specific structures that determine how they work. This folding process is a delicate dance of physics and chemistry.

Understanding protein structure and folding is crucial for grasping how life works at the molecular level. We'll explore the different levels of protein structure, from primary to quaternary, and dive into the fascinating world of protein folding mechanisms and the factors that influence this process.

Protein structure levels and features

Primary and secondary structures

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• The primary structure of a protein is the linear sequence of amino acids linked together by peptide bonds, with the specific order determined by the genetic code and unique to each protein
• The secondary structure refers to the local folding of the polypeptide chain into regular structures stabilized by hydrogen bonds between amino acid residues
  • Alpha helices are right-handed coiled structures with 3.6 amino acid residues per turn, where the backbone carbonyl oxygen of each amino acid forms a hydrogen bond with the backbone amino hydrogen of the amino acid located four residues ahead in the sequence (e.g., keratin)
  • Beta sheets are formed by extended polypeptide chains arranged either parallel or antiparallel to each other, with hydrogen bonds forming between the backbone carbonyl oxygen and amino hydrogen of adjacent polypeptide strands (e.g., silk fibroin)

Tertiary and quaternary structures

• The tertiary structure is the three-dimensional arrangement of a single polypeptide chain, resulting from the folding and spatial organization of secondary structures, and stabilized by various noncovalent interactions (hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions)
  • The tertiary structure is critical for the biological function of a protein, as it determines the specific shape and surface properties (e.g., enzymes, antibodies)
• Quaternary structure refers to the assembly of two or more polypeptide chains (subunits) into a multi-subunit complex, held together by the same types of noncovalent interactions that stabilize tertiary structure
  • Quaternary structure is a feature of multi-subunit proteins, but not all proteins have this level of organization (e.g., hemoglobin, DNA polymerase)

Thermodynamics of protein folding

Thermodynamic principles and driving forces

• Protein folding is a spontaneous process driven by the laws of thermodynamics, particularly the second law, which states that systems tend to progress towards a state of maximum entropy (disorder)
• The native (folded) state of a protein is the thermodynamically most stable conformation, as it minimizes the free energy of the system, which is determined by the balance between enthalpy and entropy
  • Enthalpy refers to the heat content of the system and is influenced by the formation of noncovalent interactions (hydrogen bonds, van der Waals forces) during protein folding
  • Entropy is a measure of the disorder or randomness of the system, and protein folding results in a decrease in entropy, as the unfolded state has more conformational freedom than the folded state
• The hydrophobic effect plays a crucial role in protein folding, with nonpolar amino acid residues clustering in the interior of the protein, away from the aqueous environment, to minimize their contact with water molecules and reduce the overall free energy of the system

Energy landscape and folding intermediates

• The folding process often involves the formation of intermediate states, which are partially folded conformations with local energy minima
• The energy landscape of protein folding is typically described as a funnel-shaped surface, with the native state at the bottom of the funnel representing the global energy minimum
  • The funnel shape illustrates the progressive narrowing of conformational space as the protein approaches its native state
  • Folding intermediates may represent kinetic traps or productive on-pathway states, depending on their stability and the energy barriers separating them from the native state

Chaperones in protein folding

Chaperone functions and families

• Molecular chaperones are proteins that assist in the folding of other proteins, particularly during protein synthesis or under stress conditions (heat shock), helping to prevent protein misfolding and aggregation
• Chaperones can be classified into different families based on their structure and function
  • Hsp60 chaperones (GroEL in bacteria) form large, barrel-shaped complexes that encapsulate unfolded or partially folded proteins, providing a favorable environment for folding
  • Hsp70 chaperones bind to exposed hydrophobic regions of unfolded or misfolded proteins, preventing aggregation and promoting proper folding
  • Hsp90 chaperones are involved in the maturation and activation of specific client proteins, such as steroid hormone receptors and protein kinases
• Chaperones work in concert with co-chaperones, which regulate their activity and specificity (e.g., Hsp40 and nucleotide exchange factors in the Hsp70 system)

Quality control and other chaperone roles

• In addition to their role in protein folding, chaperones are involved in protein quality control, targeting misfolded or damaged proteins for degradation through the ubiquitin-proteasome system or autophagy
• Chaperones also participate in the assembly of multi-subunit protein complexes and the transport of proteins across membranes
  • Examples include the translocation of proteins into mitochondria or the endoplasmic reticulum
  • Chaperones like Hsp70 and Hsp90 assist in the assembly of the 26S proteasome and the folding of proteins in the endoplasmic reticulum (e.g., BiP)

Kinetics and pathways of protein folding

Experimental techniques and folding rates

• Protein folding is a complex process that occurs on a timescale ranging from microseconds to seconds or even minutes, with the folding rate depending on factors such as protein size, sequence, and the presence of chaperones
• The folding process can be studied using various experimental techniques
  • Stopped-flow fluorescence spectroscopy measures changes in intrinsic fluorescence of tryptophan residues during folding
  • Circular dichroism monitors the formation of secondary structures (alpha helices and beta sheets)
  • NMR spectroscopy provides residue-specific information on the formation of tertiary structure
• These methods allow researchers to monitor the formation of secondary and tertiary structures in real-time and characterize folding intermediates

Folding models and transition state analysis

• Protein folding pathways can be described using different models
  • The framework model proposes that secondary structures form independently and then assemble into the final tertiary structure
  • The nucleation-condensation model suggests that the formation of a folding nucleus, consisting of a few key residues, is the rate-limiting step in protein folding, with the rest of the protein rapidly condensing around it once the nucleus is formed
• Phi-value analysis is a powerful method for studying the transition state of protein folding by introducing specific mutations and measuring their effects on folding rates, allowing researchers to infer the structure of the transition state ensemble
  • A phi-value of 1 indicates that the mutated residue is fully structured in the transition state, while a phi-value of 0 suggests that the residue is unstructured
  • Fractional phi-values indicate partial structure formation in the transition state

Misfolding and aggregation

• Misfolding and aggregation of proteins can lead to the formation of amyloid fibrils, which are associated with various neurodegenerative diseases (Alzheimer's, Parkinson's)
  • Amyloid fibrils are composed of cross-beta structures, where beta strands from multiple protein molecules form intermolecular hydrogen bonds perpendicular to the fibril axis
  • Amyloid formation typically involves a nucleation-dependent polymerization process, with a lag phase followed by rapid fibril growth
• Understanding the kinetics and mechanisms of protein misfolding is crucial for developing therapeutic strategies against these diseases
  • Inhibiting the initial nucleation event or stabilizing the native state of the protein are potential approaches to prevent amyloid formation
  • Chaperones and small molecules that modulate protein folding and aggregation are being explored as therapeutic agents