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Hydrogen bonds or ‘H-bonds’ are polar, non-covalent bonds or interactions between a hydrogen atom (H) attached to a more electronegative atom, such as oxygen (O) or nitrogen (N), which partially pulls the electron cloud away from the H, leaving it electropositive—with another electronegative atom, such as O or N from a different molecule or from a different part of the same molecule. H-bond interactions play a huge role in the biochemistry of living processes, and in the structures and interactions of biological molecules, with each other and with different molecules including water. Nature's natural solvent, water, is itself a dynamic H-bonded polar structure, which strongly affects solubility and, as (dynamic) water of hydration, interactions between molecules. Compared with covalent and ionic bonds, H-bonds are individually much weaker (

We recall the experimental approaches involved in the discovery of hydrogen bonds in deoxyribonucleic acid (DNA) made 70 years ago by a team of scientists at University College Nottingham led by J.M. Gulland, and in relation to previous studies. This discovery proved an important step in the elucidation of the correct structure for DNA made by J.D. Watson and F.H.C. Crick, as acknowledged in ‘The Double Helix’. At that time of the discovery, however, it was impossible to delineate between inter- and intra-chain hydrogen bonds. We also consider in the light of more recent hydrodynamic theory a tentative model for DNA proposed by Gulland's and D.O. Jordan's PhD student J.M. Creeth in his PhD thesis of 1948, with the correct prediction of two chains with a sugar-phosphate backbone on the exterior and hydrogen-bonded bases between the nucleotide bases of opposite chains in the interior. Our analysis shows that his incorporation of alternating breaks in the two-chain structure was not necessary to explain the viscosity data on scission of hydrogen bonds after titrating to high or low pH. Although Creeth's model is a depiction of DNA structure alone, he could not know whether the hydrogen bonding was intermolecular, although this was subsequently proved correct by others. The mechanisms by which replicative processes occurred were of course unknown at that time, and so, he could not have realised how closely his tentative model resembled steps in some viral replicative mechanisms involving the molecule of life that he was working on.

We describe the detailed biochemical characterization of CYP74C3 (cytochrome P450 subfamily 74C3), a recombinant plant cytochrome P450 enzyme with HPL (hydroperoxide lyase) activity from Medicago truncatula (barrel medic). Steady-state kinetic parameters, substrate and product specificities, RZ (Reinheitszahl or purity index), molar absorption coefficient, haem content, and new ligands for an HPL are reported. We show on the basis of gel filtration, sedimentation velocity (sedimentation coefficient distribution) and sedimentation equilibrium (molecular mass) analyses that CYP74C3 has low enzyme activity as a detergent-free, water-soluble, monomer. The enzyme activity can be completely restored by re-activation with detergent micelles, but not detergent monomers. Corresponding changes in the spin state equilibrium, and probably co-ordination of the haem iron, are novel for cytochrome P450 enzymes and suggest that detergent micelles have a subtle effect on protein conformation, rather than substrate presentation, which is sufficient to improve substrate binding and catalytic-centre activity by an order of magnitude. The kcat/Km of up to 1.6×108 M−1·s−1 is among the highest recorded, which is remarkable for an enzyme whose reaction mechanism involves the scission of a C–C bond. We carried out both kinetic and biophysical studies to demonstrate that this effect is a result of the formation of a complex between a protein monomer and a single detergent micelle. Association with a detergent micelle rather than oligomeric state represents a new mechanism of activation for membrane-associated cytochrome P450 enzymes. Highly concentrated and monodispersed samples of detergent-free CYP74C3 protein may be well suited for the purposes of crystallization and structural resolution of the first plant cytochrome P450 enzyme.

Adenosylcobalamin-dependent glutamate mutase (EC 5.4.99.1) from Clostridium tetanomorphum comprises two protein components, MutE and MutS. The formation of the holoenzyme is a kinetically complex process that involves the co-operative association of MutS, MutE and adenosylcobalamin. The MutS portion of the cobalamin-binding site is conserved within a group of adenosylcobalamin-dependent enzymes that catalyse similar isomerizations. However, in contrast with glutamate mutase, in these other enzymes the cobalamin-binding region represented by MutS is present as a C-terminal domain. We have investigated the effect on the structural and kinetic properties of glutamate mutase of linking MutS to the C-terminus of MutE. Kinetic analysis of this protein, MutES, showed, unexpectedly, that enzyme activity was still co-operatively dependent on protein concentration. The Km for l-glutamate was unchanged from the wild type, whereas Vmax was decreased to approx. one-thirtieth and the Km for coenzyme increased approx. 10-fold. Investigation of the quaternary structure of MutES by equilibrium ultracentrifugation indicated that the protein existed in equilibrium between monomeric and dimeric forms. Thus linking MutE and MutS together seems to substantially weaken the contacts that are responsible for the dimerization of MutE. The two domains of the MutES monomer seem unable to communicate, so that active enzyme is formed by the intermolecular association of two MutES subunits in a co-operative manner.

A large proportion of adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermannii is isolated in an inactive form which contains a tightly bound cobalamin. Even when the enzyme was denatured in 5.0 M guanidine hydrochloride the cobalamin remained associated with the protein. However, when dithiothreitol was added to the denatured protein, the pink inhibitor was rapidly converted into a yellow-brown compound which could be removed by dialysis. Enzyme activity could be recovered after removal of the denaturant, although surprisingly this did not depend on prior treatment with dithiothreitol. The interaction between the protein and inhibitor was investigated by using analytical ultracentrifugation under denaturing conditions. The sedimentation coefficient s20,w was measured in various concentrations of guanidine hydrochloride. A complicated picture emerged in which at low denaturant concentrations subunit dissociation, partial unfolding and aggregation occur, whereas at high concentration the protein behaves as a monodisperse species. No major differences in sedimentation were observed between the enzyme-cobalamin complex and the cobalamin-free enzyme, suggesting that the inhibitor does not significantly stabilize higher-order structure within the protein.

This paper compares the biophysical and mechanistic properties of a typical type I dehydroquinase (DHQase), from the biosynthetic shikimate pathway of Escherichia coli, and a typical type II DHQase, from the quinate pathway of Aspergillus nidulans. C.d. shows that the two proteins have different secondary-structure compositions; the type I enzyme contains approx. 50% alpha-helix while the type II enzyme contains approx. 75% alpha-helix. The stability of the two types of DHQase was compared by denaturant-induced unfolding, as monitored by c.d., and by differential scanning calorimetry. The type II enzyme unfolds at concentrations of denaturant 4-fold greater than the type I and through a series of discrete transitions, while the type I enzyme unfolds in a single transition. These differences in conformational stability were also evident from the calorimetric experiments which show that type I DHQase unfolds as a single co-operative dimer at 57 degrees C whereas the type II enzyme unfolds above 82 degrees C and through a series of transitions suggesting higher orders of structure than that seen for the type I enzyme. Sedimentation and Mr analysis of both proteins by analytical ultracentrifugation is consistent with the unfolding data. The type I DHQase exists predominantly as a dimer with Mr = 46,000 +/- 2000 (a weighted average affected by the presence of monomer) and has a sedimentation coefficient s0(20,w) = 4.12 (+/- 0.08) S whereas the type II enzyme is a dodecamer, weight-average Mr = 190,000 +/- 10,000 and has a sedimentation coefficient, s0(20,w) = 9.96 (+/- 0.21) S. Although both enzymes have reactive histidine residues in the active site and can be inactivated by diethyl pyrocarbonate, the possibility that these structurally dissimilar enzymes catalyse the same dehydration reaction by the same catalytic mechanism is deemed unlikely by three criteria: (1) they have very different pH/log kcat. profiles and pH optima; (2) imine intermediates, which are known to play a central role in the mechanism of type I enzymes, could not be detected (by borohydride reduction) in the type II enzyme; (3) unlike Schiff's base-forming type I enzymes, there are no conserved lysine residues in type II amino acid sequences.

On 19 and 20 June 2003, a joint meeting was held at the University of Nottingham between the Biochemical Society (as part of their Focused Meetings series) and the British Biophysical Society, which focused on the molecular interactions. Interactions between molecules underpin the whole of biological science, both in 2D, as in membrane systems, and in ‘3D’, or aqueous systems.

In the colon there is acontinuous, insoluble adherent mucus gel covering the surface mucosa and forming a protective barrier. The gel forming properties of the mucus barrier are dependent on the component mucins. Mucin subunits consisting of a central protein core with glycosylated and non-glycosylated regions are joined together by disulphide bridges (between the non-glycosylated regions) to form polymers.(~)Following exhaustive proteolysis only the glycosylated region of the subunit remains intact. The polymeric structure of mucin is essential for it's gel forming and viscous properties.@) At present 7 mucin genes have been partially characterised, 6 of which are secreted mucins (MUC 2-7).(3.4)Sequencing has essentially been confined to the major glycosylated region of the mucin core which consists of tandemly repeated amino acid sequences. For each MUC gene the number of tandem repeats varies as does the number andcomposition of aminoacidscharacteristic ofthese tandem repeats. Although all the tandem repeats are rich in threonine and to a lesser extentserine, theproportionofthreoninetoserinevaries. MUC 3,4,5,6 & 7 have thr:ser ratios of 1.4: I , 1.3: 1,2: I , 1.8: 1 & 1.4: 1 respectively, , whereas serine is absent from MUC2. At least three of these genes, MUC 2,3 &4, have been shown tobeexpressedin thecolon. MUC 2 (which is expressed in the colon) is the best characterised mucin gene, it's sequence being essentially complete apart from the amino terminal.(5*6)It comprises -4700 aa and has two glycosylated repetitive regions of different molecular size; a23 aa tandem repeat region of -23OOaa which is very rich in thr, 60% of the amino acids, and a347 aa base pair repeat region containing 49% thr and 10% ser. Cys rich regions are located both at the carboxyl and amino ends of the molecule. This study set out to investigate the structure of human and pig colonic mucin isolated from the secreted adherent mucus gel and to reconcile these structures with the identified geneproducts. Glycoprotein from pig and human colonic mucus was extracted in proteinase inhibitors and purified by equilibrium centrifugation in 3.5M CsCl followed by gel filtration on Sepharose CL-2B. Reduced glycoprotein was produced by reduction with mercaptoethanol, 24 hr, 20oC; proteolytically digested mucin by digestion with papain 0 . 0 8 ~ mg-lmucin, 60OC, 48 hr.The molecular weight of mucin was determined by sedimentation equilibrium, using a Beckman Model E analytical ultracentrifuge, and multi-angle light scattering. Dilute solution viscosity measurements were made using aCouette rotating vis~ometerat25~C.Thiol analysis was by the methodofMantleetaf. ( I 990)(6) Purified mucin was reduced with 0.33M NaBH,, thiol groupslabelledwith4,4dithyopyridine.The number oftotal and free thiols were calculated using the molar absorption coefficient. Amino ac id a n a l y s i s w a s by the me thod of Car l ton and Morgan ( 1 988).(7)Samples hydrolysedfor24 hr at 1 10°C were derivatised with 9 fluorenylmethylchloroformate and analysed by reverse phase HPLC. Purified mucin glycoproteins were largely excluded (60%) from Sepharose CL-2B. Reduction of mucin produced a broad single peak which was also mainly excluded. Digestion produced a smaller species includedon SepharoseCL-2B (Kav.0.56). The weight average molecular weight of polymeric and digested mucin was determined by two methods, sedimentation equilibrium and light scattering. Measurements from both methods were in close agreement. The M,forpolymeric and digested mucin weredetermined to be5.7x1O6and5.5xlO5respectively. The papaindigested mucin was asingle sizedpolydisperse species. The weight average molecular weight of reduced mucin determined by light scattering was 3 . 3 ~ 106. This size distribution was confirmed by intrinsic viscosity studies. Polymeric, reduced and digested mucin having intrinsic viscosity values of0.27,O. 10 and0.02 mg ml-'respectively . Polymeric colonic mucincontained 35 23.1 nmol mg-1 total thiols including 7.0 nmol mg-'free thiol groups. Reduced much contained 23i4Snmolmg-1 total thiolsincluding 17nmolmg-1 free thiolgroups. The majority of thiols in polymeric colonic mucin -28 nmol mg-lare involved in disulphide bridge formation. This together with the large number present andthedecreasein molecular sizeon reductionof the isolated mucin is consistent with their involvement in polymerisation. The presence of -3 nmol mg-I disulphide bridges after reduction suggests the presence of intra-molecular disulphide bridges possibly involved in a globular protein structure and presumeably inacessible to the reducing agent. Proteolysis removed all thiols suggesting that they are confined to the non-glycosylated region of the protein core acessible to the proteolytic enzyme. Both human and pig colonic mucin contained a high proportion of ser, thrandpro,atotalof39% and40% by weightoftotalproteincontent respectively. Digestion of pig colonic mucin resulted in loss of -30% of protein by weight from 17% to 12% of the total molecule. Thr and ser were conserved which is characteristic of the glycosylated regions of other mucins.(')The ratio of thr:ser in papain digested humanandpigmucin was-1 : 1. From these results pig colonic much isolated in the presence of proteolytic inhibitors has amolecularweightof5.7~ 106andcontains -14 nmol mg-I disulphide bridges (ie 84disulphide bridgesperpolymer). Eachpolymerconsistsof at least 2 subunits Mr3.3x106linked by disulphide bridges. Each subunit contains 4-6 similar size regions of Mr 5 . 5 ~ 105. The glycosylated region of both human and pig mucin is rich in thr, ser and pro with a thr:ser ratio of -1 : 1. Proteolysis removes all the non-glycosylated part of the mucin to leave the heavily glycosylated protected protein core which presumeably represents the tandem repeat segment of the mucin. Therefore i t is interesting to compare the amino acid analysis of the digested mucins with the published sequences of tandem repeats of the MUC genes. The amino acid analysis for both human and pig glycosylated region has a much higher ser content relative to thr than would be expected for the tandem repeat region ofMUC 2&3, expressed in the human colon. The thr:ser ratio is most compatible with that of the tandem repeat region of MUC4 which has a thr:ser ratio 1.2: I , however the total amino acid composistion of humandigestedmucin isdifferenttothatoftttthe tandem repeat region of MUC4. Further the size of theglycosylated region and the presence of asingle species does notequate with MUC 2. This analysis described here for the secreted colonic mucin is not compatible with the products of the m u c h genes so far sequenced being the major component of the secreted adherent colonic mucus gel.

The effect of increasing ionic strength on adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii was studied by using analytical ultracentrifugation. Both sedimentation-velocity and low-speed sedimentation-equilibration measurements show that the enzyme dissociates progressively into its two dissimilar subunits with increasing ionic strength. Equilibrium between the alpha beta-dimer and the separated subunits is rapidly established under these conditions. Dissociation is accompanied by loss of enzymic activity, but the position of the equilibrium is unaffected by the presence of either substrate or adenosylcobalamin cofactor.

We have purified to homogeneity the regions derived by chymotryptic digestion of the ox neurofilament polypeptides NFH and NFM; the regions, called M1 and M2, are thought to form part of the projecting sidearms of mammalian neurofilaments [Chin, Eagles & Maggs (1983) Biochem. J. 215, 239-252]. They were isolated and purified under non-denaturing conditions and showed no tendency to interact with each other in solution. The Mr values obtained by sedimentation are approx. 61,000 for M1 and 42,000 for M2, considerably lower than the values obtained by SDS/polyacrylamide-gel electrophoresis. These Mr values were unchanged in the presence of 6 M-guanidine hydrochloride, suggesting that the regions exist as monomers in solution. Both M1 and M2 are highly phosphorylated, and there is only a slight change in the sedimentation value upon dephosphorylation. Dephosphorylation of M1 with alkaline phosphatase was more than 90% efficient but was never absolute. Dephosphorylation of M2 was complete. Both M1 and M2 bind Ca2+; in the case of M1, this binding is phosphorylation-dependent. M1 also binds cytochrome c, and dephosphorylation affects binding. In similar conditions, neurofilaments bind at least twice their own mass of cytochrome c, owing to their opposite net charges. No interactions were observed between native or dephosphorylated M1 and M2, and intact neurofilaments under a wide variety of conditions. These results are discussed in terms of the possible roles that neurofilament sidearms might play and throw doubt upon their supposed function of rigidly cross-linking neurofilaments together within the axoplasm of neurons.

Theories concerning the concentration-dependence of sedimentation and diffusion coefficients for macro-molecules in dilute solution are compared and discussed, together with their experimental basis. An attempt has been made to clarify an important uncertainty still present in the literature as to whether sedimentation coefficients should be corrected for solvent or solution density. It is pointed out that the two processes yield the same extrapolation limit but different concentration-dependencies, which have, however, been related. A general expression is derived thermodynamically for the concentration-dependence of diffusion that includes the coefficient of the concentration term involved in sedimentation (on the basis of sedimentation coefficients corrected from solution density). For rigid spherical particles the expression is shown to be exactly equivalent to one given by Batchelor [(1976) J. Fluid Mech. 74, 1-29], which was derived on the basis of sedimentation coefficients corrected from solvent density. Finally, we discuss the concentration-dependence of apparent weight-average relative molecular masses (‘molecular weights’) (from, e.g., sedimentation equilibrium) and note an important omission in some earlier representations.

The flexible and greatly expanded roughly spherical model for mucus glycoproteins proposed earlier, on the basis of hydrodynamic and n.m.r. data, is supported by new hydrodynamic results on a bronchial glycoprotein from a cystic-fibrosis patient. Furthermore, images from electron microscopy of this molecule and a lower-molecular-weight mucus glycoprotein (which closely resembles a glycopolypeptide) appear to be at least consistent with this model.

A new hydrodynamic shape function, lambda, is derived for determining the conformation of biological macromolecules in solution, adding to the increasing number of shape parameters whose experimental determination does not require a knowledge of the particle swelling due to solvation in solution. lambda can be found from a knowledge of the molecular weight, intrinsic viscosity and the harmonic mean rotational relaxation time. A table of values and a plot of lambda as a function of axial ratio for both oblate and prolate ellipsoids of revolution are given.

A simple check for the presence of heterogeneity in a macromolecular system is proposed, employing comparison of Rayleigh sedimentation-equilibrium patterns for two solutions of the same fringe concentration but differing absolute concentrations. The method is illustrated by application to a bronchial glycoprotein from a cystic-fibrosis patient.

Fluorescence Fig. 1. Histogram of spot fluorescent signal; artefacts have been distingiiished from spots as described in the text (u) For six fibroblasts treated with Dil-labelled LDL. The continuous line is the best fit of the log-normal distribution found in (h) for single particles, convolved to give up to five particles per spot. (h) For DiI-LDL on a polylysine-coated slide. The continuous line is the best fit of a log-normal distribution.