Random coil polypeptide linear

Figure 2. Gel permeation data for polypeptide linear random coils plotted according to the method of Porath (8) M0,555 is plotted vs. Kd1/3. Lines drawn through the data from each column are lines of best fit determined by linear least-squares analysis. Numerical designation for each curve represents the agarose...

Gel permeation chromatography of protein linear random coils in guanidinium chloride allows simultaneous resolution and molecular weight analysis of polypeptide components. Column calibration results are expressed in terms of a log M vs. Kd plot or of effective hydrodynamic radius (Re/). For linear polypeptide random coils in 6M GuHCl, Re is proportional to M0 555, and M° 555 or Re may be used interchangeably. Similarly, calibration data may be interpreted in terms of N° 555 (N is the number of amino acid residues in the polypeptide chain), probably the most appropriate calibration term provided sequence data are available for standards. Re for randomly coiled peptide heteropolymers is insensitive to amino acid residue side-chain composition, permitting incorporation of chromophoric, radioactive, and fluorescent substituents to enhance detection sensitivity. 

Previous studies (5-7) have clearly demonstrated that gel permeation chromatography of reduced linear randomly coiled polypeptide chains in GuHCl provides an accurate, dependable method for molecular weight estimation. 

Figure 3. Gel permeation data for linear randomly coiled polypeptides on various agarose resins, plotted according to the method of Ackers (9). M0 555 is plotted vs. the inverse error function complement of Kd (erfc 1 Kd). Lines drawn through the data points represent best fits obtained from linear least-squares analysis of the data. Numerical designation of each curve represents the percent agarose composition for the resin used. Filled triangles on the curve for the 6% resin, and the filled squares on the curve for the 10% resin are points determined using fluorescent proteins. Data for the labeled polypeptides were not included in the least-squares analysis.

Gel permeation studies on agarose-GuHCl columns provide for high resolution and accurate molecular weight determination of linear randomly coiled polypeptide chains. For nonlinear random coils, gel permeation studies provide for accurate determination of the effective hydrodynamic radius of the components. 

Current investigations on dilute polymer solutions are still largely limited to the class of macromolecular solutes that assume randomly coiled conformation. It is therefore natural that there should be a growing interest in expanding the scope of polymer solution study to macromolecular solutes whose conformations cannot be described by the conventional random-coil model. The present paper aims at describing one of the recent studies made under such impetus. It deals with a nonrandom-coil conformation usually referred to as interrupted helix or partial helix. This conformation is a hybrid of random-coil and helix precisely, a linear alternation of randomly coiled and helical sequences of repeat units. It has become available for experimental studies through the discovery of helix-coil transition phenomena in synthetic polypeptides. 

Since this article is limited to linear polymers, and since polyelectrolytes have been essentially excluded, there are relatively few biochemically interesting materials which we can discuss. Several polypeptides, however, can be treated by the present methods. It is now well known that many polypeptides can exist in two widely different conformations in solution, the random coil and the a-helix. This was first... 

The origin of nonzero bo values in these cases is not entirely clear, but it can be formally traced to the difference between Xo and values for the simple dispersion of random coils and hence to a failure of the assumption that Xo equals Xo. If K equals Xo, then the first term of the Moffitt equation will of course be the same as the simple Drude expression known to describe the data and, there being no necessity for a second term, bo will vanish. However, if Xo differs from Xo, the Moffitt plot may still be linear but with a nonvanishing slope. Thus dispersion data that are simple when referred to one dispersion constant may appear complex when plotted against another by a form that sees matters as complex, thereby generating what may be properly suspected as pseudocomplexity. The Moffitt equation was initially intended to describe the complex dispersion of polypeptides for which the simple Drude equation is inadequate, but, as will be seen, its form is also applied to protein dispersions which can be expressed equally well by either formula. It is therefore important to examine more fully the relation of the two equations for cases in which both fit the data. 

The complexity and diversity of structures in the native proteins eluded any attempt to produce some simple conformation that accounted for their interfacial properties. The study of synthetic polypeptides with non-polar side chains has provided good evidence to support the view that the a-helix can be stable at the air-water interface (5), and it is therefore possible that the interfacial denaturation of proteins is mainly a loss of the tertiary structure (6, 7, 8). Since for a typical protein an a-helix takes up about the same area per residue as the p conformation, it can be accommodated as easily. Moreover, like the p conformation but unlike a more randomly coiled structure, it is linear and therefore compatible with a plane surface without loss of configurational entropy (5). In this respect a plane surface may favor an ordered over a more random structure. The loss of solubility of the spread protein can then be attributed to intermolecular association between hydrophobic side chains exposed as a result of the action of the interface on the polar exterior of the molecules. 

The systems studied by these authors are poly-L-glutamate in aqueous 0.3 ikf sodium phosphate at pH 7.85 and 37°, poly-L-lysine in aqueous 1.0 M sodium bromide at pH 4.54 and 37°, (and poly- -benzyl-E-aspartate in w-cresol at 100°). The tendency for these polypeptides to form ordered structures limits the choice of solvent systems in which random coil dimensions may be studied. These solvents, furthermore, cannot be solvents for the randomly coiled form of the polypeptides. However, if conditions are achieved (like those under which Brant and Flory carried out their investigation), such that the linear expansion of... 

Before proteins can actively function in the living cell they must fold up into a specific 3-dimensional structure, the so-called native state (see Fig. 1). Already in the 1960 s it was recognized that the long linear polypeptides chains can adopt their native structure starting from the random coil state in a surprisingly short time. The famous Levinthal paradox states that if a peptide bond between amino acids can only adopt two conformations a relatively short protein of a hundred residues can have around 2 10 possible... 

All enzyme molecules possess the primary, secondary, and tertiary structural characteristics of proteins (see Chapter 20). In addition, most enzymes also exhibit the quaternary level of structure. The primary structure, the linear sequence of amino adds linked through their a-carboxyl and a-amino groups by peptide bonds, is specific for each type of enzyme molecule. Each polypeptide cham is coiled up into three-dimensional secondary and tertiary levels of structure. Secondary structure refers to the conformation of limited segments of the polypeptide chain, namely a-helices, P-pleated sheets, random coils, and p-turns. The arrangement of secondary structural elements and amino acid side chain interactions that define the three-dimensional structure of the folded protein is referred to as its tertiary structure. In many cases biological activity, such as the catalytic activity of enzymes, requires two or more folded polypeptide chains (subunits) to associate to form a functional molecule. The arrangement of these subunits defines the quaternary structure. The subunits may be copies of the... 

To allow for an access of two anchor groups to two identical or different active sites from the non-primed S subsites, the crystal structure of Ac-Leu-Leu-Nle-H bound to pS and pS of the yeast 20S proteasome was used as a template [34]. The entry of substrates into the proteolytic chamber is restricted by the bottle-neck of the a ring, which recruits from outside only fully unfolded linear polypeptides for digestion. This fact significantly restricts the choice of spacers for bivalent inhibitor constructs. Such a spacer should mimic as much as possible the unstructured polypeptide chain of an unfolded protein, and reach a length of about 50 A. Peptides of appropriate size are known to be rapidly degraded by the yeast proteasome, and thus linear polyoxyethylene (PEG) chains were selected as mimic of random-coiled polypeptide chains [37, 64], since this polymer is known to be highly solvated and... 

The investigations presented focus on interpretation of polarization of fluorescence measurements and use of these measurements to study the structure of a representative spectrum of linear synthetic polypeptides, a vinyl polymer, and an intramolecularly cross-linked synthetic polypeptide. The methodological studies investigate the validity of the transition temperature as a structural parameter, the interaction of the fluorescent dye and the polymer to which it is conjugated, and the influence of the dye-polymer interaction on the measurements of various molecular parameters. The structural studies focus on the structure of the random coil, the helix-coil transition, the a-helix to conformation transition in polylysine, and the stability of the spatial structure in intramolecularly cross-linked synthetic polypeptides. 

The problem of the sense of the helix has also been attacked with the aid of optical rotation measurements in the following manner. Downie et al. (1957) investigated the optical rotation of copolymers of l- and D-leucine in benzene and in trifluoroacetic acid. In the latter solvent the copolymers exist as random coils, independent of the fraction of the L-form [designated as l/(d -f- l)]. Since the residue rotation (corrected for the dispersion of the refractive index) is a linear function of l/(d - - l), as shown in Fig. 96b, and is zero when l/(d + l) = 0.5, the observed rotation may be attributed to the excess of l- over n-residues. This conclusion is based on the assumption that the optical rotation of independent groups is additive, and is supported by the straight-line graphs obtained for a variety of solvents and randomly coiled polypeptides. The existence of the polypeptide in the randomly coiled form in trifluoroacetic acid is consistent with the absence of a contribution from any helical configuration. 

Both the structural and functional properties of a protein depend ultimately on the nature and sequence of amino acids in the polypeptide chain or chains of which it is composed. This is referred to as its primary structure and the peptide bonds that join the various amino acids together are strong covalent bonds. The primary structure of a protein may be compared with the structural formula of a small molecular compound, but protein chains do not usually exist either in a simple extended linear form or as a random coil. Instead they have a specific three-dimensional structure which is predetermined by the primary structure and which, in turn, determines their biological characteristics. 

With Brownian dynamics it is possible to simulate the dynamics of short stretches of amino acid sequence that form secondary structure to observe multiple transitions between secondary structure and random coil confonnations in one trajectory. In most Brownian liynamics studies, the polypeptide chain is modeled as a linear chain of suitably parameter-ized spheres, with each sphere representing an amino acid residue. Thus, whereas only times of the order of nanosec-onds can be achieved in atomic-detail MD simulations, times up to a microsecond can be simulated with Brownian dynamics using this simplified model of the protein. Both helix-coil" and )8-sheet -coil transitions have been studied by means of Brownian dynamics. Ccnnputed rate constants for both transitions are of the order of tens of reciprocal nanoseconds in both directions, and are consistent with available experimental data. 

Proteins have complex molecular structures. The linear sequence of the amino acids comprising a protein is classified as its primary structure. In different proteins, these linear sequences assume conserved structures along the axis of the polypeptide in the form of alpha-helixes, 3j -helix, beta sheets, or random coils, turns which are described as the secondary structure of the protein. These secondary structures are stabilized primarily by hydrogen bonds. For thermodynamic stability, proteins rearrange themselves into tertiary structures comprising several secondary structures stabilized by van der Waal s, electrostatic, or hydrophobic interactions, hydrogen bonding, as well as disulfide cross-links. Some proteins have a fourth structural level called the quaternary structure in which two or more... 

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