BCH Graduate Survey of Biochemistry

Transcription

1 BCH 5045 Graduate Survey of Biochemistry Instructor: Charles Guy Producer: Ron Thomas Director: Glen Graham Lecture 7 Slide sets available at:

2 David L. Nelson and Michael M. Cox LEHNINGER PRINCIPLES OF BIOCHEMISTRY Fifth Edition CHAPTER 3 Amino Acids, Peptides and Proteins 2008 W. H. Freeman and Company

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4 You should know names, 3-letter and 1-letter abbreviations for the protein amino acids

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6 Why bother thinking of grouping amino acids and especially this way?

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8 Watch this video and see if you can pick up on a vast overstatement:

9 Only two amino acids have strong absorbance properties in the UV range around 280 nm. If you use absorbance to measure protein, these are the amino acids that provide the absorbancy. There is a problem with this approach to quantifying proteins by absorbance. What is it?

10 Cysteine and Cystine have a thiol group (reduced) and a disulfide bond (oxidized) respectively.

11 Here are some uncommon amino acids found in cells.

12 Pyrrolysine HC N H H 2 C C C N CH 2 CH 2 CH 2 CH 2 CH COO C H H O NH H 2 3 C Selenocysteine (Sec) and pyrrolysine (Pyl), are the 21 st and 22 nd protein amino acids, and they are encoded by UGA and UAG, respectively, which are codons that usually function as stop signals.

13 Fig. 1. Structures of pyrrolysine derivatives Ambrogelly et al. (2004) Proc. Natl. Acad. Sci. USA 101, Copyright 2004 by the National Academy of Sciences Pyrrolysine, the 22nd cotranslationally inserted amino acid, was found in the Methanosarcina barkeri monomethylamine methyltransferase protein in a position that is encoded by an in-frame UAG stop codon in the mrna. M. barkeri encodes a special amber suppressor trna (trnapyl) that presumably recognizes this UAG codon. It was reported that Lys-tRNAPyl can be formed by the aminoacyl-trna synthetase-like M. barkeri protein PylS [Srinivasan, G., James, C. M. & Krzycki, J. A. (2002) Science 296, ], whereas a later article showed that Lys-tRNAPyl is synthesized by the combined action of LysRS1 and LysRS2, the two different M. barkeri lysyl-trna synthetases. Pyrrolysyl-tRNAPyl formation was presumed to result from subsequent modification of lysine attached to trnapyl. To investigate whether pyrrolysine can be directly attached to trnapyl we chemically synthesized pyrrolysine. We show that PylS is a specialized aminoacyl-trna synthetase for charging pyrrolysine to trnapyl; lysine and trnalys are not substrates of the enzyme. In view of the properties of PylS we propose to name this enzyme pyrrolysyl-trna synthetase. In contrast, the LysRS1:LysRS2 complex does not recognize pyrrolysine and charges trnapyl with lysine. These in vitro data suggest that Methanosarcina cells have two pathways for acylating the suppressor trnapyl. This would ensure efficient translation of the inframe UAG codon in case of pyrrolysine deficiency and safeguard the biosynthesis of the proteins whose genes contain this special codon.

14 Important naturally occurring non-protein amino acids found in organisms.

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17 Here we see our first peptide or in this case a tetrapeptide.

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20 Protein structure

21 Because some amino acids have positive or negatively charged sidechains, then proteins containing these amino acids will have an overall charge. The net charge of a protein is determined by the relative contents of the charged amino acids. Over a ph range, proteins will have a net positive or negative charge except where the number of positive charged sidechains exactly equals the number of negative charged sidechains, then it will have a zero net charge and the ph where the net charge is zero is known as the isoelectric point pi. Gel electrophoresis as shown on the left takes advantage of the fact that placing a charged protein in an electrical field in a gel matrix will cause the protein to migrate towards an electrode. If the net charge on the protein is negative, it will migrate towards the positive electrode. In the example on the left, proteins would be separated on the basis of their net charge and molecular weight or size. However, on the right, if the proteins are first coated with a detergent having massive amounts of negative charge, then the separation that occurs is largely dependent on the molecular radius or more generally, the molecular weight of the protein. You are seeing on the right the actual proteins after they have been stained for visualization.

22 PATRICK O FARRELL developed the 2-D gel electrophoresis of proteins technique when he took advantage of the fact that a protein will migrate in an electric field until it reaches its isoelectric point and then stop if there is an immobilized ph gradient in the gel matrix. So he separated proteins on the basis of their charge, rotated the orientation of separation 90, then separated them on the basis of size. The paper where he described the technique in 1975 has been cited more than 14,000 times.

23 Frederick Sanger Proteins are made from individual amino acid building blocks. By the 1950s this had been long known from the analyses of protein hydrolyzates that yielded free amino acids. While overall composition of proteins is important, what really defines the potential function of proteins is the sequence of amino acids in the protein. Interview with Fred Sanger if you are interested

24 Christian Anfinsen would later win the Nobel Prize in Chemistry in 1972 for his studies on the relationship between protein sequence, folding and function. Anfinsen s and the work of others established that all the information necessary for a protein to fold into its biologically functional 3-dimensional structure was encoded in the amino acid sequence. Anfinsen s work rested squarely on the discoveries of Frederick Sanger, the recipient of the 1958 Nobel Prize in chemistry, for his work on the structure of proteins, particularly insulin. Sanger believed that protein sequence was the key to understanding living matter and set out to determine the exact sequence of amino acids in insulin. He developed the reagent, dinitrofluorobenzene (FDNB), that reacted with amino groups present in proteins to form an acid-stable dinitrophrenyl (DNP) derivative. The DNP protein was treated with acid to break the polypeptide backbone, and the free DNP amino acid derivatives were isolated and compared to known amino acids standards.

25 How Sanger did it. Sanger s work would ultimately lead to the use of recombinant insulin, for example: Generic Name: glargine which is produced by recombinant DNA technology in a nonpathogenic strain of Escherichia coli (K12) as the production organism. Glargine differs from human insulin in that amino acid asparagine at position A21 is replaced by glycine and two arginines have been added to the C-terminus of the B-chain.

26 The Sanger FDNB method for sequencing a protein was laborious and time consuming. Pehr Edman developed a cycling process using phenylisothiocyanate (PITC) which reacts with the amino group at the amino terminus under basic conditions to form a phenylthiocarbamyl derivative (PTC-protein). Trifluoroacetic acid is used to cleave off the derivatized N-terminal amino acid as its anilinothialinone derivative(atz-amino acid) leaving a new amino terminal amino acid for the next cycle of the degradation process cycle. The ATZ amino acid is then removed by extraction with ethyl acetate and converted to a phenylthiohydantoin derivative (PTH-amino acid).

27 The PTH-amino acid is chromatographed through a HPLC reversedphase C-18 column for detection at 270 nm. A standard mixture of 19 PTH-amino acids is chromatographed as the first cycle of the sequencing run. This chromatogram provides standard retention times of the amino acids for comparison with each Edman degradation cycle. The HPLC chromatograms are collected using a computer data analysis system. This process is repeated sequentially to provide up to 30 amino acid residues from the N- terminal sequence of the protein/peptide.

28 For all proteins made on and by ribosomes, their sequences are determined by the nucleotide sequences in the cell s genome(s) embodied in the genetic code(s) as illustrated above. Understanding how this all occurs is perhaps one of the greatest achievements of humankind and a great story as well.