Protein essential residue determination

Determinations of Numbers and Types of Essential Residues. Determining the number and types of essential residues in proteins has been approached by a number of methods (I). The simplest, but perhaps... 

RNA directs biosynthesis of various peptides and proteins essential for any living organisms. Protein biosynthesis seems to be catalysed by mRNA rather than protein-based enzymes and occur on the ribosome. On the ribosome, the mRNA acts as a template to pass on the genetic information that it has transcribed from the DNA. The specific ribonucleotide sequence in mRNA forms an instruction or codon that determines the order in which different amino acid residues are to be joined. Each instruction or codon along the mRNA chain comprises a sequence of three ribonucleotides that is specific for a given amino acid. For example, the codon U-U-C on mRNA directs incorporation of the amino acid phenylalanine into the growing protein. 

Carboxypeptidases are zinc-containing enzymes that catalyze the hydrolysis of polypeptides at the C-terminal peptide bond. The bovine enzyme form A is a monomeric protein comprising 307 amino acid residues. The structure was determined in the laboratory of William Lipscomb, Harvard University, in 1970 and later refined to 1.5 A resolution. Biochemical and x-ray studies have shown that the zinc atom is essential for catalysis by binding to the carbonyl oxygen of the substrate. This binding weakens the C =0 bond by... 

Both types of mutations have been made in T4 lysozyme. The chosen mutations were Gly 77-Ala, which caused an increase in Tm of 1 °C, and Ala 82-Pro, which increased Tm by 2 °C. The three-dimensional structures of these mutant enzymes were also determined the Ala 82-Pro mutant had a structure essentially identical to the wild type except for the side chain of residue 82 this strongly indicates that the effect on Tm of Ala 82-Pro is indeed due to entropy changes. Such effects are expected to be additive, so even though each mutation makes only a small contribution to increased stability, the combined effect of a number of such mutations should significantly increase a protein s stability. 

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