Sequencing, protein

In this chapter, the strategies for identifying the amino acid sequence in a polypeptide chain are outlined. The amino acid composition of a protein, i.e. the relative abundance of each amino acid in the peptide chain is often a first step 

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The sequence space of proteins is extremely dense. The number of possible protein sequences is 20. It is clear that even by the fastest combinatorial procedure only a very small fraction of such sequences could have been synthesized. Of course, not all of these sequences will encode protein stmctures which for functional purjDoses are constrained to have certain characteristics. A natural question that arises is how do viable protein stmctures emerge from the vast sea of sequence space The two physical features of folded stmctures are (l)in general native proteins are compact but not maximally so. (2) The dense interior of proteins is largely made up of hydrophobic residues and the hydrophilic residues are better accommodated on the surface. These characteristics give the folded stmctures a lower free energy in comparison to all other confonnations. 

Analysis of tlie global statistics of protein sequences has recently allowed light to be shed on anotlier puzzle, tliat of tlie origin of extant sequences [170]. One proposition is tliat proteins evolved from random amino acid chains, which predict tliat tlieir length distribution is a combination of the exponentially distributed random variable giving tlie intervals between start and stop codons, and tlie probability tliat a given sequence can fold up to fonii a compact... 

The protein sequence database is also a text-numeric database with bibliographic links. It is the largest public domain protein sequence database. The current PIR-PSD release 75.04 (March, 2003) contains more than 280 000 entries of partial or complete protein sequences with information on functionalities of the protein, taxonomy (description of the biological source of the protein), sequence properties, experimental analyses, and bibliographic references. Queries can be started as a text-based search or a sequence similarity search. PIR-PSD contains annotated protein sequences with a superfamily/family classification. 

PIR National Biomedical Research Form-dation protein sequence biblio., sub- stance, se- quence 280 000 protein sequences journals, author submis- sions National Biomedical Research Foundation free periodi- cally http //pir.geor- getown.edu... 

Fig. 10.22 Hidden Markov model used for protein sequence analysis, are match states (corresponding in this...

Fig, 10.25 The six environment categories used by the 3D profiles method. (Figure adapted from Bowie j U, R Liith. and D Eisenberg 1991. A Method to Identify Protein Sequences That Fold into a Known Three-Dunensinnal Structure. Science 253 164-170.)... 

Gap A break in a DNA or protein sequence which enables two or more sequence to be aligned... 

PIR-International Protein Sequence Database Redundant protein sequence database... 

Swiss-Prot, TrEMBL Annotated non-redundant protein sequence database, TrEMBL is a computer-annotated supplement to Swiss-Prot. TrEMBL contains the translations of all coding sequences present in the EMBL Nucleotide Sequence Database which are no yet integrated into Swiss-Prot... 

Each element of matrix corresponds to the probability that the amino acid in coiumn wiii mutate to the jmino acid in row j after a period of 1 PAM. The vaiues have been multipiied by 10000. (Based on Dayhoff M O 1978. Atlas of Protein Sequence and Structure Voiume 5 Suppiement 3. Dayhoff M O (Editor) Georgetown Jniversity Medicai Center, National Biomedical Research Foundation Figure 82.)... 

Barton G J1996. Protein Sequence Alignment and Database Scanning. In Sternberg M E (Editor) Prote Structure Prediction - A Practical Approach. Oxford, IRL Press, pp. 31-63. 

Barton G J 1998. Protein Sequence Aligrunent Techniques. Acta Crystallographica 054 1139-1146. Blundell T L, B L Sibanda, M J E Sterbnerg and J M Thornton. Knowledge-based Prediction of Prote Structures and the Design of Novel Molecules. Nature 326 347-352. 

Bryant S H and C E Lawrence 1993. An Empirical Energy Function for Threading Protein Sequences Through the Folding Motif. Proteins Structure, Punction and Genetics 16 92-112. 

ChemSketch has some special-purpose building functions. The peptide builder creates a line structure from the protein sequence defined with the typical three-letter abbreviations. The carbohydrate builder creates a structure from a text string description of the molecule. The nucleic acid builder creates a structure from the typical one-letter abbreviations. There is a function to clean up the shape of the structure (i.e., make bond lengths equivalent). There is also a three-dimensional optimization routine, which uses a proprietary modification of the CHARMM force field. It is possible to set the molecule line drawing mode to obey the conventions of several different publishers. 

The amount of sample required is quite small as little as 10 mole is typical So many peptides and proteins have been sequenced now that it is impossible to give an accurate count What was Nobel Prize winning work m 1958 is routine today Nor has the story ended Sequencing of nucleic acids has advanced so dramatically that it is possible to clone the gene that codes for a particular protein sequence its DNA and deduce the structure of the protein from the nucleotide sequence of the DNA We 11 have more to say about DNA sequencing m the next chapter... 

Section 28 11 Three RNAs are involved m gene expression In the transcription phase a strand of messenger RNA (mRNA) is synthesized from a DNA tern plate The four bases A G C and U taken three at a time generate 64 possible combinations called codons These 64 codons comprise the genetic code and code for the 20 ammo acids found m proteins plus start and stop signals The mRNA sequence is translated into a prescribed protein sequence at the ribosomes There small polynucleotides called... 

The importance of linked scanning of metastable ions or of ions formed by induced decomposition is discussed in this chapter and in Chapter 34. Briefly, linked scanning provides information on which ions give which others in a normal mass spectrum. With this sort of information, it becomes possible to examine a complex mixture of substances without prior separation of its components. It is possible to look highly specifically for trace components in mixtures under circumstances in which other techniques could not succeed. Finally, it is possible to gain information on the molecular structures of unknown compounds, as in peptide and protein sequencing (see Chapter 40). 

The "overlap" information allows the original protein sequence to be generated. In this example, the sequence D-E-F-G in the fourth peptide proves that the first and second peptides must have been joined originaliy at E,F and not A,H. 

Protein sequencing by creation of overlap peptides (mapping),... 

Kinter, M. and Sherman, N.E., Protein Sequencing and Identification Using Tandem Mass Spectrometry, Wiley, Chichester, U.K., 2000. 

PVDF-based microporous filters are in use at wineries, dairies, and electrocoating plants, as well as in water purification, biochemistry, and medical devices. Recently developed nanoselective filtration using PVDF membranes is 10 times more effective than conventional ultrafiltration (UF) for removing vimses from protein products of human or animal cell fermentations (218). PVDF protein-sequencing membranes are suitable for electroblotting procedures in protein research, or for analyzing the phosphoamino content in proteins under acidic and basic conditions or in solvents (219). 

A potentially general method of identifying a probe is, first, to purify a protein of interest by chromatography (qv) or electrophoresis. Then a partial amino acid sequence of the protein is deterrnined chemically (see Amino acids). The amino acid sequence is used to predict likely short DNA sequences which direct the synthesis of the protein sequence. Because the genetic code uses redundant codons to direct the synthesis of some amino acids, the predicted probe is unlikely to be unique. The least redundant sequence of 25—30 nucleotides is synthesized chemically as a mixture. The mixed probe is used to screen the Hbrary and the identified clones further screened, either with another probe reverse-translated from the known amino acid sequence or by directly sequencing the clones. Whereas not all recombinant clones encode the protein of interest, reiterative screening allows identification of the correct DNA recombinant. 

As described earlier, translation of the EPSPS mRNA of plants results in the formation of a protein which has an AJ-terminal extension. The AJ-terminal extension, referred to as the chloroplast transit peptide, is necessary and sufficient for the import of the preprotein by the chloroplast. Once imported by the chloroplast, the transit peptide is cleaved releasing the mature enzyme. As expected, introduction of the EPSPS transit peptide to other protein sequences results in the importation of the fusion protein by the chloroplast. 

Fig. 10. The receptor—G-protein sequence. An activated receptor interacts with the trimeric GDP-ligated receptor to cause an interchange of GDP by GTP and dissociation into the activated Ga—GTP (left) and G y (right) subunits. These then interact with a variety of effectors. The purpose of the activated...

Although the techniques described have resulted in the determination of many protein stmctures, the number is only a small fraction of the available protein sequences. Theoretical methods aimed at predicting the 3-D stmcture of a protein from its sequence therefore form a very active area of research. This is important both to understanding proteins and to the practical appHcations in biotechnology and the pharmaceutical industries. 

W. Machleidt, Proc. Int. Conf. So/idPhase Methods in Protein Sequence Anal. 17 (1975). 

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