The relationship between molecular weight and quaternary structure of globular proteins

The relationship between the molecular weight and the number of subunits in oligomeric globular proteins consisting of identical subunits has been analyzed. It has been shown that the molecular weights of the subunits are distributed about a mean value of 48,000 and consequently that the molecular weights of the native oligomeric proteins are distributed in clearly distinguishable molecular weight regions. This observation allows the probability of a particular oligomeric structure to be predicted from a measurement of the oligomer molecular weight alone, which is useful in a number of types of study of protein structure, particularly comparative studies. Calculations have been performed which suggest that there is no thermodynamic limitation, in terms of the subunit interactions themselves, to the size of an oligomeric protein with a given number of subunits. Rather, an individual polypeptide chain itself has inherent size limitations, which consequently limits the molecular weight of the corresponding oligomer.     

Topology of globular proteins

This paper inquires whether it is reasonable to expect the native structure of proteins to be “knotted”. To this end, some topological properties of polypeptides containing disulfide bridges are discussed using notions from mathematical knot theory and graph theory. The probability of occurrence of knots in random cyclic polymers is calculated as a function of chain length by elementary Monte Carlo methods. The implications of this for protein renaturation and for determining the tertiary structure of proteins are discussed.     

Thermodynamics of globular proteins

The analysis of temperature-induced unfolding of proteins in aqueous solutions was performed. Based on the data of thermodynamic parameters of protein unfolding and using the method of semi-empirical calculations of hydration parameters at reference temperature 298 K, we obtained numerical values of enthalpy, free energy, and entropy which characterize the unfolding of proteins in the ‘gas phase’. It was shown that specific values of the energy of weak intramolecular bonds (?H^int), conformational free energy (?G^conf) and entropy (?S^conf) are the same for proteins with molecular weight 7–25 kDa. Using the energy value (?H^int) and the proposed approach for estimation of the conformational entropy of native protein (S^NC), numerical values of the absolute free energy (G^NC) were obtained.     

Analysis of the relationship between side-chain conformation and secondary structure in globular proteins

The relationship between the preferred side-chain dihedral angles and the secondary structure of a residue was examined. The structures of 61 proteins solved to a resolution of 2.0 A (1 A = 0.1 nm) or better were analysed using a relational database to store the information. The strongest feature observed was that the chi 1 distribution for most side-chains in an alpha-helix showed an absence of the g- conformation and a shift towards the t conformation when compared to the non-alpha/beta structures. The exceptions to this tendency were for short polar side-chains that form hydrogen bonds with the main-chain which prefer g+. Shifts in the chi 1 preferences for residues in the beta-sheet were observed. Other side-chain dihedral angles (chi 2, chi 3, chi 4) were found to be influenced by the main-chain. This paper presents more accurate distributions for the side-chain dihedral angles which were obtained from the increased number of proteins determined to high resolution. The means and standard deviations for chi 1 and chi 2 angles are presented for all residues according to the secondary structure of the main-chain. The means and standard deviations are given for the most popular conformations for side-chains in which chi 3 and chi 4 rotations affect the position of C atoms.     

Conformational stability of globular proteins

The conformational stability of ribonuclease T1 has been measured as a function of the variables of most interest to biochemists: temperature, pH, salt concentration, disulfide-bond content and amino acid sequence. The results provide insight into the forces that stabilize globular proteins.     

The adsorption of globular proteins

A series of experiments is suggested to elucidate further the nature of the adsorption of globular proteins on polar solid surfaces. Two ratios of surface energies and the total protein volume are used to characterize the expected form of the adsorbed species. A simple model calculation illustrates the approach.     

Foam fractionation of globular proteins

Foam fractionation of bovine serum albumin (BSA) was studied as a model system for potato wastewater. The effects of feed concentration, superficial gas velocity, feed flow rate, bubble size, pH, and ionic strength on the enrichment and recovery of BSA were investigated in a single-stage continuous foam fractionation column. Enrichments ranged from 1.5 to 6.0 and recoveries from 5 to 85%. The feed concentrations were varied from 0.01 to 0.2 wt %, and enrichments were found to increase with lower feed concentrations. Enrichments also increased with lower superficial gas velocities and larger bubble sizes. At sufficiently low feed flow rates, enrichment was found to increase with an increase in the flow rate, eventually becoming insensitive to the feed flow rate at higher values. The pH was varied from 3.5 to 7.0 and ionic strength from 0.001M to 0.2M. The effects of pH and ionic strength were found to be coupled with bubble size. A minimum bubble size was found at pH 4.8, the isoelectric point of BSA, resulting in a minimum in the enrichment. Bubble size, and thus enrichment, was found to increase as the ionic strength decreased from 0.2M to 0.01M. Previous models(1,2) for the hydrodynamics of foam column were extended for a singlestage continuous foam fractionation column for the prediction of enrichment and recovery. The model assumed adsorption equilibrium, infinite surface viscosity, and bubbles of the same size. Though coalescence was formally accounted for in the model by considering bubble size as a function of foam height, calculations for the experimental runs were performed only for the case of no coalescence. Quantitative predictions of enrichment and recovery could not be made with a single representative bubble size because of the broad inlet bubble size distribution as well as broadening of the distribution as a result of coalescence. The experimental enrichments were higher and recoveries were lower than the model predictions, the discrepancy being more pronounced at lower feed concentrations because of increased coalescence. The higher enrichments are due to the predominant effect of internal reflux as a result of coalescence whereas the lower recoveries are a result of detrimental effects of broadening bubble size distributions.     

Salting-in characteristics of globular proteins

Protein solubility, and the formation of various solid phases, is of interest in both bioprocessing and the study of protein condensation diseases. Here we examine the the phase behavior of three proteins (chymosin B, β-lactoglobulin B, and pumpkin seed globulin) previously known to display salting-in behavior, and measure their solubility as a function of pH, ionic strength, and salt type. Although the phase behavior of the three proteins is quantitatively different, general trends emerge. Stable crystal nucleation does not occur within the salting-in region for the proteins examined, despite the crystal being observed as the most stable solid phase. Instead, two types of amorphous phases were found within the salting-in region; additionally, an analog to the instantaneous clouding curve was observed within the salting-in region for chymosin B. Also, protein solutions containing sulfate salts resulted in different crystal morphologies depending on whether Li₂SO₄ or (NH₄)₂SO₄ was used.     

Nanofibers made of globular proteins

Strong nanofibers composed entirely of a model globular protein, namely, bovine serum albumin (BSA), were produced by electrospinning directly from a BSA solution without the use of chemical cross-linkers. Control of the spinnability and the mechanical properties of the produced nanofibers was achieved by manipulating the protein conformation, protein aggregation, and intra/intermolecular disulfide bonds exchange. In this manner, a low-viscosity globular protein solution could be modified into a polymer-like spinnable solution and easily spun into fibers whose mechanical properties were as good as those of natural fibers made of fibrous protein. We demonstrate here that newly formed disulfide bonds (intra/intermolecular) have a dominant role in both the formation of the nanofibers and in providing them with superior mechanical properties. Our approach to engineer proteins into biocompatible fibrous structures may be used in a wide range of biomedical applications such as suturing, wound dressing, and wound closure.     

Thermal stabilities of globular proteins

Statistical thermodynamic theory has recently been developed to account for the stabilities of globular proteins. Here we extend that work to predict the dependence on temperature. Folding is assumed to be driven by solvophobic interactions and opposed by the conformational entropy. The temperature dependence of the solvophobic interaction is taken from the transfer experiments on amino acids by Tanford and Nozaki and on model solutes by Gill and Wads?. One long-standing puzzle has been why proteins denature upon heating, since the solvophobic force to fold strengthens with increasing temperature. This is resolved by the theory, which predicts two first-order phase transitions. "Cold denaturation" is driven principally by the weakening of the solvophobic interaction, but normal denaturation is driven principally by the gain of conformational entropy of the chain. Predictions of the thermodynamic state functions are in reasonable agreement with the calorimetric experiments of Privalov and Khechinashvili. Comparison of the theory with experiments suggests that there may be an additional enthalpic driving force toward folding which is not due to the solvophobic interactions.     

Disulphide bridges in globular proteins

Disulphide bridges in proteins of known sequence, connectivity and structure were studied to search for common features. Their distribution, topology, conformation and conservation were analysed in detail. Several general patterns emerge which to some extent dictate disulphide bridge formation. For example, there is a strong preference for shorter connections, with half-cystines separated by less than 24 residues in 49% of all disulphides. Right- and left-handed disulphides occur equally; the left-handed structures adopt one predominant conformation (symmetric χ₁ = ?60 °, χ₂ = ?80 °, χ₃ = t-90 °). Cystines are generally very well conserved, in contrast to cysteines, with a free —SH group, which mutate rapidly. If a disulphide is not conserved, both cystines are mutated. The role of disulphide bridges in globular proteins is discussed.     

Origins of globular structure in proteins

Thermodynamic incompatibility of polymers in a common solvent is possibly a driving force for formation and evolution of globular protein structures. Folding of polypeptide chains leads to a decrease in both excluded volume of molecules and chemical differences between surfaces of globular molecules with chemical information hidden in the hydrophobic interior. Folding of polypeptide chains results in 'molecular or thermodynamic mimicry' of globular proteins and in at least more than 10-fold higher phase separation threshold values of mixed protein solutions compared to those of classical polymers. Unusually high co-solubility might be necessary for efficient biological functioning of proteins, e.g. enzymes, enzyme inhibitors, etc.     

Origins of globular structure in proteins

Since natural proteins are the products of a long evolutionary process, the structural properties of present-day proteins should depend not only on physico-chemical constraints, but also on evolutionary constraints. Here we propose a model for protein evolution, in which membranes play a key role as a scaffold for supporting the gradual evolution from flexible polypeptides to well-folded proteins. We suggest that the folding process of present-day globular proteins is a relic of this putative evolutionary process. To test the hypothesis that membranes once acted as a cradle for the folding of globular proteins, extensive research on membrane proteins and the interactions of globular proteins with membranes will be required.     

Far-infrared spectra of some globular proteins

The far-infrared absorption spectrum (40–400 cm^?1) of solid pellets and films of several globular proteins (lysozyme, myoglobin, hemoglobin, serum albumin, ribonuclease, chymotrypsinogen, subtilisin) and of some representative polypeptides [nylon 66, poly (γ-benzyl L -glutamate)] have been investigated by using a Michelson interferometer. While polypeptides are known to present several peaks which can be assigned mostly to hydrogen-bond modes, all the investigated globular proteins display only one broad, intense baud in the 100–200 cm^?1 region. The origin of this band, which persists even after denaturation or partial digestion, is discussed.     

Loop fold nature of globular proteins

Protein chains make numerous returns in globules, thus forming loops, closed by tight residue-to-residue contacts-closed loops. Previous statistical analysis of the sizes and locations of the closed loops in all major protein folds revealed that the loops have an almost standard contour length of 25-30 amino acid residues and follow one after another along the chain. In this work the closed loops of the major folds are presented in three dimensions. A special image filtering procedure is introduced that allows one to visualize the standard size closed loops for the first time. The loop positions along the sequences are verified by detection of loop-end clusters.