Small proteins

Ortiz A R, A Kolinski and J Skolnick 1998. Fold Assembly of Small Proteins Using Monte C Simulations Driven by Restraints Derived from Multiple Sequence Alignments. Jourru Molecular Biology 277 419-446. 

Eleven chirality centers may seem like a lot but it is nowhere close to a world record It is a modest number when compared with the more than 100 chirality centers typ ical for most small proteins and the thousands of chirality centers present m nucleic acids A molecule that contains both chirality centers and double bonds has additional opportunities for stereoisomerism For example the configuration of the chirality center m 3 penten 2 ol may be either R or S and the double bond may be either E or Z There fore 3 penten 2 ol has four stereoisomers even though it has only one chirality center... 

Fig. 5. Protein folding. The unfolded polypeptide chain coUapses and assembles to form simple stmctural motifs such as -sheets and a-hehces by nucleation-condensation mechanisms involving the formation of hydrogen bonds and van der Waal s interactions. Small proteins (eg, chymotrypsin inhibitor 2) attain their final (tertiary) stmcture in this way. Larger proteins and multiple protein assembhes aggregate by recognition and docking of multiple domains (eg, -barrels, a-helix bundles), often displaying positive cooperativity. Many noncovalent interactions, including hydrogen bonding, van der Waal s and electrostatic interactions, and the hydrophobic effect are exploited to create the final, compact protein assembly. Further stmctural...

Polypeptides. These are a string of a-amino acids usually with the natural 5(L) [L-cysteine is an exception and has the R absolute configuration] or sometimes "unnatural" 7f(D) configuration at the a-carbon atom. They generally have less than -100 amino acid residues. They can be naturally occurring or, because of their small size, can be synthesised chemically from the desired amino acids. Their properties can be very similar to those of small proteins. Many are commercially available, can be custom made commercially or locally with a peptide synthesiser. They are purified by HPLC and can be used without further purification. Their purity can be checked as described under proteins. 

With mostly unambiguous data, this protocol has been successfully used for proteins with up to 160 residues [62]. Although virtually all structures converge to the correct fold for small proteins, we observe that approximately one-third of the structures are misfolded for larger proteins, or for low data density, or many ambiguities (see, e.g.. Ref. 63). We have also used this protocol for most structure calculations with the automated NOE assignment method ARIA discussed in the next section. 

Standard calculation methods developed for small proteins are sufficiently powerful to solve protein structures and complexes in the 30 kDa range and beyond [97,98] and protein-nucleic acid complexes [99]. Torsion angle dynamics offers increased conver-... 

Although comparative modeling is the most accurate modeling approach, it is limited by its absolute need for a related template structure. For more than half of the proteins and two-thirds of domains, a suitable template structure cannot be detected or is not yet known [9,11]. In those cases where no useful template is available, the ab initio methods are the only alternative. These methods are currently limited to small proteins and at best result only in coarse models with an RMSD error for the atoms that is greater than 4 A. However, one of the most impressive recent improvements in the field of protein structure modeling has occurred in ab initio prediction [155-157]. 

AR Ortiz, A Kolinski, J Skolnick. Fold assembly of small proteins using Monte Carlo simulations driven by restraints derived from multiple sequence alignments. J Mol Biol 277 419-448, 1998. 

T Dandekar, P Argos. Folding the mam chain of small proteins with the genetic algorithm. J Mol Biol 236 844-861, 1994. 

Figure 2.19 Organization of polypeptide chains into domains. Small protein molecules like the epidermal growth factor, EGF, comprise only one domain. Others, like the serine proteinase chymotrypsin, are arranged in two domains that are required to form a functional unit (see Chapter 11). Many of the proteins that are involved in blood coagulation and fibrinolysis, such as urokinase, factor IX, and plasminogen, have long polypeptide chains that comprise different combinations of domains homologous to EGF and serine proteinases and, in addition, calcium-binding domains and Kringle domains.

Small protein modules form adaptors for a signaling network... 

To obtain the secondary and tertiary stmcture, which requires detailed information about the arrangement of atoms within a protein, the main method so far has been x-ray crystallography. In recent years NMR methods have been developed to obtain three-dimensional models of small protein molecules, and NMR is becoming increasingly useful as it is further developed. 

Figure 18.11 Electron-density maps at different resolution show more detail at higher resolution, (a) At low resolution (5.0 A) individual groups of atoms are not resolved, and only the rodlike feature of an

From a map at low resolution (5 A or higher) one can obtain the shape of the molecule and sometimes identify a-helical regions as rods of electron density. At medium resolution (around 3 A) it is usually possible to trace the path of the polypeptide chain and to fit a known amino acid sequence into the map. At this resolution it should be possible to distinguish the density of an alanine side chain from that of a leucine, whereas at 4 A resolution there is little side chain detail. Gross features of functionally important aspects of a structure usually can be deduced at 3 A resolution, including the identification of active-site residues. At 2 A resolution details are sufficiently well resolved in the map to decide between a leucine and an isoleucine side chain, and at 1 A resolution one sees atoms as discrete balls of density. However, the structures of only a few small proteins have been determined to such high resolution. 

Figure 18.16 One-dlmenslonal NMR spectra, (a) H-NMR spectrum of ethanol. The NMR signals (chemical shifts) for all the hydrogen atoms In this small molecule are clearly separated from each other. In this spectrum the signal from the CH3 protons Is split Into three peaks and that from the CH2 protons Into four peaks close to each other, due to the experimental conditions, (b) H-NMR spectrum of a small protein, the C-terminal domain of a cellulase, comprising 36 amino acid residues. The NMR signals from many individual hydrogen atoms overlap and peaks are obtained that comprise signals from many hydrogen atoms. (Courtesy of Per Kraulis, Uppsala, from data published in Kraulis et al.. Biochemistry 28 7241-7257, 1989.)...

In NMR the magnetic-spin properties of atomic nuclei within a molecule are used to obtain a list of distance constraints between those atoms in the molecule, from which a three-dimensional structure of the protein molecule can be obtained. The method does not require protein crystals and can be used on protein molecules in concentrated solutions. It is, however, restricted in its use to small protein molecules. 

Eleven chirality centers may seem like a lot, but it is nowhere close to a world record. It is a modest number when compared with the more than 100 chirality centers typical for most small proteins and the thousands of chirality centers present in nucleic acids. 

Finnt, D. F., et al., 1987. Tandem qnadrnpole Fourier tran.sform ma.ss. spectrometry of oligopeptides and small protein.s. Proceedings of the National Academy of Sciences, U.S.A. 84 620—623. 

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