protein denaturation and Anfinsen's dogma

what is protein denaturation?

process where protein loses 3D shape, making it biologically inactive

what are the consequences of loss of 3D shape

loss of structure

loss of function

what causes protein denaturation?

external stresses break the bonds that maintain the protein’s structure (affects secondary, tertiary and quaternary structure)

primary sequence of amino acids remains intact

how does temperature denature a protein

• increase temperature - molecular motion increase, hydrogen bonds disrupted

• decrease temperature - ice forms, protein structure breaks

how do changes in pH denature proteins?

• Alter protonation state of certain R groups

• Alters hydrogen bonding and salt bridge patterns

how do reducing agents denature proteins?

• E.g. b-mercaptoethanol and dithiothreitol

• Convert disulfide bridges to sulfhydryl groups

what is Anfinsen’s dogma?

dogma states that states that a protein's amino acid sequence completely determines its native, functional three-dimensional structure

how did Anfisen demonstrate this dogma?

• denatured ribonuclease A (single polypeptide chain protein) with urea and β-mercaptoethanol (2ME) which breaks disulfide bonds

• he then removed the urea and 2ME via dialysis

• the polypeptide spontaneously refolded correctly, reformed its disulfide bonds and its catalytic activity was restored

what happened when 2ME was removed first and urea after

• when 2ME was removed first and urea second, the protein only regained 1% of its original catalytic activity

• cysteine residues formed disulfide bonds randomly showing that weak bonding interactions are required for correct positioning of disulfide bonds