Allosteric formulation of thermal transitions in macromolecules, including effects of ligand binding and oligomerization

We examine the effects of concentration (aggregation), buffers, and ligation, under conditions of either constant ligand activity or limited total amount of ligand, upon thermal denaturation of macromolecules as measured by scanning calorimetry. In doing so we utilize and extend an earlier generalized allosteric treatment [S. J. Gill, B. Richey, G. Bishop, and J. Wyman (1985) Biophys. Chem. 21, 1-14], applicable to ligand binding, enthalpy changes, and volume changes in a macromolecular system. The approach is contrasted with formulations based on the idea of structural domains. We show how information from the full scanning calorimetric curves can be utilized in arriving at and testing appropriate models for observed behavior in selected examples.     

Statistical mechanical deconvolution of thermal transitions in macromolecules. III. Application to double-stranded to single-stranded transitions of nucleic acids

The statistical mechanical deconvolution theory for macromolecular conformational transitions is extended to the case of nucleic acids transitions involving strand separation. It is demonstrated that the partition function, Q, as well as all the relevant thermodynamic quantities of the system, can be calculated from experimental scanning calorimetric data. In particular, it is shown that important thermodynamic parameters such as the size of the average cooperative unit during strand separation, the mean helical segment length, and the mean coil-segment length can be calculated from the average excess enthalpy function 〈ΔH〉. The theory is applied to the double-stranded to single-stranded transition of the system poly(A)·poly(U) at different salt concentrations. It is shown that the mean helical segment length is a monotonically decreasing function of the temperature well before strand separation occurs. On the other hand, the mean coil segment length remains practically constant until temperatures very close to T_m. Both experimental findings clearly indicate that the unfolding of poly(A)·poly(U) proceeds through the formation of many short helical sequences. The cooperative unit for the strand separation is calculated to be about 120 base pairs and essentially independent of the salt concentration. The existence of a minimum helical segment length of 10 ± 2 base pairs within the double-stranded form is calculated.     

Local dynamics in biological macromolecules

The diffusive approach in the optimized Rouse-Zimm approximation to segment relaxation in the nanosecond time domain (ORZLD) is extended to consider chains of nonequivalent units as occurring in biological macromolecules. The correlation times for second-order time correlation functions of each virtual bond on the chain are calculated for some homopolypeptides, and random and regular copolypeptides. The expected correlation times for biological macromolecules organized in multiple domains are discussed via a simple model of the ORZLD hierarchy. Dynamic bond correlation times are compared with static local persistence lengths.     

Microcalorimetry of biological macromolecules

The capabilities of contemporary differential scanning and isothermal titration microcalorimetry for studying the thermodynamics of protein unfolding/refolding and their association with partners, particularly target DNA duplexes, are considered. It is shown that the predenaturational changes of proteins must not be ignored in studying the thermodynamics of formation of their native structure and their complexes with partners, particularly their cognate DNA duplexes.     

Light-scattering from detergent-complexed biological macromolecules

A theory is described for Rayleigh light-scattering from solutions of detergent-complexed macromolecules applicable to measurements carried out under conditions of Donnan equilibrium. The theory shows that when scattering measurements are made on detergent-solubilized macromolecules in the presence of detergent micelles the apparent Mr is dependent on the extent of detergent binding and effective charge on the detergent-macromolecule complex and the micellar charge and aggregation number. Equations are given for the apparent Mr of the macromolecule under limiting conditions of high salt and low salt concentration. Low-angle laser-light-scattering measurements were made on lysozyme complexed with sodium n-dodecyl sulphate both in the absence and in the presence of detergent micelles. These experimentally obtained data were used in conjunction with the detergent-binding isotherm to test the theory at high ionic strength. Light-scattering measurements were also made on detergent-saturated complexes as a function of ionic strength and pH. The results are in reasonable accord with both the qualitative and the quantitative predictions of the theory.     

The cybernetics of biological macromolecules

This paper develops the concept of linkage as it applies to the binding, of ligands by a polyfunctional macromolecule, or system of macromolecules, under equilibrium, steady-state, and transient conditions.     

Self-assembly of biological macromolecules.

The genetic apparatus of the cell is responsible for the accurate biosynthesis of the primary structure of macromolecules which then spontaneously fold up and, in certain circumstances, aggregate to yield the complex tertiary and quaternary structures of the biologically active molecules. Structures capable of self-assembly in this range from simple monomers through oligomers to complex multimeric structures that may contain more than one type of polypeptide chain and components other than protein. It is becoming clear that even with the simpler monomeric enzymes there is becoming clear that even with the simpler monomeric enzymes there is a kinetically determined pathway for the folding process and that a folded protein must now be regarded as the minimum free energy form of the kinetically accessible conformations. It is argued that the denatured subunits of oligomeric enzymes are likely to fold to something like their final structure before aggregating to give the native quaternary structure and the available evidence would suggest that this is so. The importance of nucleation events and stable intermediates in the self-assembly of more complex structures is clear. Many self-assembling structures contain only identical subunits and symmetry arguments are very successful in accounting for the structures formed. Because proteins are themselves complex molecules and not inelastic geometric objects, the rules of strict symmetry can be bent and quasi-equivalent bonding between subunits permitted. This possibility is frequently employed in biological structures. Conversely, symmetry arguments can offer a reliable means of choosing between alternative models for a given structure. It can be seen that proteins gain stability by growing larger and it is argued in evolutionary terms that aggregation of subunits is the preferred way to increase the size of proteins. The possession of quaternary structure by enzymes allows conferral of other biologically important properties, such as cooperativity between active sites, changes of specificity, substrate channelling and sequential reactions within a multi-enzyme complex. Comparison is made of the invariant subunit compositions of the simpler oligomeric enzymes with the variation evidently open to, say, the 2-oxoacid dehydrogenase complexes of E. coli. With viruses, on the other hand, the function of the quaternary structure is to package nucleic acid and, as an example, the assembly and breakdown of tobacco mosaic virus is discussed. Attention is drawn to the possible ways in which the principles of self-assembly can be extended to make structures more complicated than those that can be formed by simple aggregation of the comonent parts.     

On a possible mechanism of the effect of microwave radiation on biological macromolecules

A model that describes the dissociation of a hydrogen bond in water clusters when irradiated by an electromagnetic field in the microwave range is proposed. The model is also applicable for the case of the rupture of the covalent bond of the water molecule in a cluster. If the energy absorption occurs at the interface of water and polymer clusters (e.g., DNA and chitosan), degradation of the polymer chain is possible.     

Mechanisms common to biological macromolecules and gels

Functional Biological macromolecules arising from folding, cross-connection and solvation of long chain biopolymers forming three-dimensional networks may be regarded as Gels. Both involve identical internal competitive forces that are selectively influenced by external conditions and conspire to adjust conformations and modulate activities. In spite of important differences in size, chemical composition, polymer bonding, density and configuration, biological macromolecules indeed manifest some of the essential physical-chemical properties of gels when involved in equilibria and rate processes. This result represents a presumptive evidence for common underlying mechanisms in functional molecules and gels. Thus, the present and highly perfectible model explains why and how, depending on initial conditions, a system may respond differently to an external parameter, and similarly to different parameters. Moreover, the fact that any localized change in one of the competitive forces gives to a pressure in the system as a whole provides an explanation for the mechanism of the transmission of information.     

Evaluation of ultrafiltration membranes with biological macromolecules

Eighteen ultrafiltration membranes ranging in molecular weight cutoff ratings from 500 to 300,000 were tested with water, 0.5M NaCl solution, and, in some cases, with macromolecules and urea in a 3-in. stirred filter cell. Approximately half of the membranes showed a significant decrease in filtration rate during the first 24-hr period. The steady-state rates were less than the manufacturers' rating for about two thirds of the membranes, the discrepancy being greater for the membranes with high molecular weight cutoffs. The filtration rates were linearly dependent on applied pressure over the range at least as great as 15 to 55 psig. The rate decreased as the concentration of macromolecules such as transfer ribonucleic acid (tRNA) increased; the rate for a concentration of 3 mg tRNA/per ml was one-fourth of that observed when no tRNA was present. Some increase in rate (～33 to 50%) was obtained by increasing the stirring speed from 100 rpm to 1000 rpm. The membranes were effective for desalting and concentration of macromolecules but not for separation of large molecules from each other, such as tRNA from bovine serum albumin. Easily denatured molecules such as catalase were not deactivated by filtration at 4°C.