A naturally occurring cross-linked TIM validates these Native Structures

Triose phosphate isomerase (TIM) from the hyperthermophilic bacterium Thermotoga maritima crystallizes as a tetramer that appears to be a pair of dimers similar to those observed in crystals of other TIMs (Maes et al. 1999). The two dimers are joined by disulphide bonds between pairs of Cys142. As noticed by Gayathri et al.(2007) this linkage is not consistent with the dimer seen in crystals being the same as the dimer in solution and Gayathri et al. conclude that the "tetramerization appears to be a crystallization artifact."
However, when the TIM monomers are docked by the methods described in this blog, the pairs of Cys142 are naturally in close proximity. The native structure of TIM from T. maritima is now included in the Gallery. It can be seen that the disulfide bond is indeed a natural part of the quaternary structure and the form of dimer seen in crystals is the actual artifact.

SUPERSEDED/OBSOLETE Mirror site for scientists in China

It has come to my notice that our colleagues in China cannot access this blog but can access the Structure Gallery. Therefore I will maintain a copy of this blog and the companion Enzyme Function blog on the server used by the Gallery. Please direct any colleagues who cannot access Blogspot to
www.nativeproteins.com/blog/

Symmetry, Pseudo-symmetry and Evolution in Protein Structures

This poster was displayed at BioPhysChem 2011 at the University of Wollongong 3-6 December 2011. Reasoning from the effect of Natural Selection on symmetry, it shows examples of the arrangement of subunits in a complex and the alignment of "barrel" structures pointing towards the centre of the complex. Click here to view the poster.

Effect of Crystallization on Protein Quaternary Structure

This talk was given at BioPhysChem2011 (3-6 December at the University of Wollongong) on 6 December 2011. It deals quantitatively with the expected changes to protein structure when the protein-in-water phase is changed to a water-in-protein phase during crystallization. For the first time there is a chemical explanation of why crystal structures differ from native structures as detailed throughout this blog. Click on the title of this post or here to view the slides.

Galactose oxidase now in the Gallery

The reassembled structure of galactose oxidase has been placed in the Gallery. My earlier work on this enzyme was posted, below, on September 30, 2007. I remarked then that the docking could be improved and I have done so in preparing the structure for the Gallery. The improved structure brings the copper centres of adjacent subunits closer together and the surrounding aromatic groups into closer contact. To achieve this I have accepted that the amino-acid chain 148-153 has been displaced by crystallization. This chain links the N-terminal auxilliary domain to the catalytic domain. It is quite likely that the N-terminal domain is also slightly out of place but it does not clash significantly with other subunits. The clash of 148-153 with the symmetry-related subunit can be seen in the reassembled structure but it is an artefact of crystallization.
Galactose oxidase shares many features with other proteins in the Gallery. Like hemocyanin it has pairs of copper-histidine centres but unlike hemocyanin, the partners in each pair are in different subunits. Like hemoglobin, the metal centres are electronically linked by an extended π-electron network. Like neuraminidases, the structural motif that focusses compressive force is a "propeller" of β-sheets. Also like some neuraminidases, galactose oxidase has auxilliary domains. These may have a role in interactions with the oligo- or poly-saccharides that are the natural substrates for both enzymes.
In coming days I will modify the preprint, linked through the 30 September 2007 post, to reflect the improved structure.

Hemocyanin structure

I was curious to see whether there were any obvious similarities in the subunit interactions of hemocyanin and hemoglobin. I downloaded 1NOL.pdb of Hazes et al. (1993). They reported it as a homohexamer and so I tried to dock the structure as a compact hexamer. In the crystal the hexamer has a large central cavity, which is unsatisfactory on physical grounds (see the first post of this Blog). Docking of a hexamer was unsatisfactory but a tetramer docked very well.
I have added the tetramer to the Gallery. The implication is that the tetramer has a physiological role.
Like hemoglobin, hemocyanin has an extensive aromatic network linked to the oxygen binding sites. Unlike hemoglobin the network does not appear to link oxygen binding sites on adjacent subunits. It is also unclear how the reported reverse Bohr effect could work.

Hemoglobin of the goose.

I have placed the R state and T state of hemoglobin from the Bar-Headed Goose, Anser indicus, in the Gallery. Inositol hexaphosphate is the polyanionic effector of avian hemoglobins and I have included a molecule of inositol hexaphosphate in the polyanion binding site of the T state. The positive charges of the polyanion binding site can be visualised by following the instructions in the Gallery. It can be seen that there are more positive charges than in human hemoglobin. The number of positive charges able to interact with the inositol hexaphosphate is more than appears at first sight, because allowance must be made for the flexibility of lysine side-chains. The larger number of positive charges accords with the fact that the avian effector has more negative charges than does diphosphoglycerate, the effector in human hemoglobin.

The Gallery can be accessed by clicking on the Title of this Post.

Quaternary Isomers of Hemoglobin

Perhaps a suitable term for the T state and R state conformations of hemoglobin would be "Quaternary Isomers". I have previously described them as interchangeable by subunit exchange. Subunit exchange is geometrically equivalent to concerted rotation of each subunit about its radial axis by 120º and perhaps this would be the preferred pathway of isomerisation in nature. For an α₂β₂ tetrahedron there are 3 different quaternary isomers. For vertebrate hemoglobins, a clockwise rotation of each subunit of the R state by 120º produces the T state with a polyanion binding site. An anticlockwise rotation of each R state subunit by 120º produces an alternate T state without an intact polyanion binding site. The alternate T state probably has a minor role physiologically. In the presence of the appropriate polyanion effector the isomerisation equilibrium would shift towards the main T state.
I have placed all 3 isomers in the Gallery.

Hemoglobin poster added to F1000 poster collection

The 2008 "Haemoglobin Revisited" poster presented at WATOC 08 has been accepted into the Faculty of 1000 Poster Collection and can be accessed by clicking here. It is slightly modified from the original by inclusion of the instructions for generating the structures and by crossing out the claim that the crystal structure cannot account for the inability to form crosslinks between the N-terminals of the beta chains. The crystal structure can account for this phenomenon if it is also assumed that the N-terminals are not very mobile. On the other hand, the native structure of the T-state proposed in the poster (click on the Gallery link) can account for the crosslinking properties only if the lysine side chains are considered mobile.
In summary, the crosslinking study cannot distinguish between these opposing views of protein structure.

Dihydrodipicolinate Synthase (DHDPS)

The structure of this enzyme from Bacillus anthracis is now available in the Gallery. It is the first alpha/beta barrel native quaternary structure to be reassembled. Using this structure as a template should help in the reassembly of other alpha/beta barrel structures. There is a ragged edge on the z-axis of this structure that corresponds to an area of inter-subunit contact in the crystal. Probably this is an example of damage to the tertiary structure caused by crystallization.

Dihydrofolate reductase Type 1 tetramer

In the course of discussions about Enzyme Function arising from the companion blog, I became aware that DHFR Type 1 is described as a monomer. It is divided into two sub-domains. As this is inconsistent with the physics of catalysis (see the first post of this blog), I explored possible dimeric and tetrameric complexes of this enzyme. The tetramer docks very well. The tetrameric structure is now included in the gallery. Click on the title of this post to study the 3D structure.
I checked the sources of reports that DHFR Type 1 is a monomer and found that the direct evidence came from experiments in the absence of cofactor. I am now planning to repeat the MW determination in the presence of cofactor.

Gallery of 3D Protein Structures

I have begun a gallery of 3D structures of the proteins mentioned on this blog. The structures are rendered by the powerful Jmol applet using the same graphic interface used by the Protein Data Bank. Jmol can display spacefill, backbone or cartoon representations. Jmol can calculate and display surfaces and cavities. It can rotate the structure in any way requested by the user and responds to scripts similar to those of RASMOL and other protein display programs. Click here to enter the gallery.

Companion Blog on Enzyme Function

Today I created a companion blog to this one, dealing with the emerging non-structural evidence in support of the role of constraint in enzyme catalysis. The blog is called "Enzyme Function" and can be viewed by clicking here. The evidence is from measurements of the kinetic isotope effects in enzymic hydride transfer reactions and subsequent attempts to simulate those effects by quantum mechanics. It is found that the effects can be simulated if atoms are brought significantly closer together than their van der Waals radii would allow. In other words the observed kinetic isotope effects imply that the active site is compressed at the time of catalysis. It is the need for compression at the time of catalysis that created the need to look for rigid protein structures as described in the current blog.

Citations of the Biophysics paper that started this blog

So far there are three laboratories that cite this work. Most active is the group of Atsushi Imai in Tokyo. They cite this work as a reason for research into the mechanical properties of single protein molecules. Their citing papers have been published in 2009, 2008, 2007, 2005a, 2005b, 2004a, and 2004b.
Another group is the Yeates group at UCLA. Their 2006 paper on knots in protein folds cited my work as justifying interest in sites of protein rigidity.
A third group (Tsekova and Sakov in Sofia, Bulgaria) published work on protein adsorption in 2005 and cited my work as justifying the study of protein-protein interactions.

Immunoglobulin structure

Last year I said that a full paper detailing all the haemoglobin work would be the next project for this blog. As it happens I realised a few months ago that IgG (an immunoglobulin) is like haemoglobin in being an α₂β₂ tetramer and so I explored tetrahedral rearrangements of the published crystal structure. This project has been very successful. I will present the IgG results on this blog before I complete the larger haemoglobin document.

Rotation-translation operators.

I have changed the form of the rotation and translation operations used to present the results of my earlier studies on neuraminidases, galactose oxidase and haemoglobin. The Convention used by the International Union of Crystallographers is that the rotation should precede the translation. I have now amended the papers linked to this blog to conform with that Convention. Previously I specified the translation before the rotation. The one paper I have not yet amended is the older pdf version of the neuraminidase paper.

I just noticed tonight that some of the punctuation and special characters in my linked htm files were not shown by Internet Explorer. Sorry about that. I have fixed the problems and added a few links back to this page to aid navigation. I'm still thinking about a novel approach to writing up the haemoglobin work. I think I have a good idea. I've also noticed that the original Biophysical Journal paper (see the bottom of this blog) is getting more citations as the years pass.

The WATOC Poster released.

Click on the text to download the poster "Haemoglobin Revisited" and instructions for generating the 3-dimensional coordinates of the structures shown in the poster.
WATOC (World Association of Theoretical and Computational Chemists) was a meeting characterised by very diverse interests.
In relation to the contentious issue raised by this blog, namely, whether a molecular structure in a crystal accurately reflects the structure in solution, the speakers expressed opinions on both sides. Those presenting work with biomolecules implicity believed that the crystal structures reflected solution structures. Those working with organo-metallic complexes, for which contradictory evidence existed, were quick to state that crystal structures could differ from solution structures. Among other participants I found a reasonable willingness to accept that crystal structures could differ from solution structures.
In relation to the "Haemoglobin Revisited" poster, one scientist was concerned that the proposed R-state differed from the T-state by too great a translocation of subunits. A number encouraged me to develop my work further and I am very grateful for their support. Perhaps reflecting the interests of participants, there was a suggestion that the proposed structures be subjected to molecular dynamics simulation to improve the docking and demonstrate stability. I agree that the docking could be improved. I am not sure that the Force Fields for Molecular Dynamics are accurate enough to simulate the weak energy of association of the haemoglobin tetramer. However, if the simulation does suggest that the proposed structures are stable, that will certainly help in the presentation of the structures.

WATOC 2008 Poster

"HAEMOGLOBIN REVISITED", poster to be shown at WATOC2008, Sydney 14-19 August
A theoretical study of catalysis by soft materials, such as protein, has shown the relationship between the degree of catalysis and local transient rigidity [1]. Since the intrinsic rigidity of protein is quite low, a theoretical source of local rigidity has been found in the effects of surface tension on multi-subunit proteins [1]. This concept has already been used to generate new quaternary structures for the neuraminidases and galactose oxidase [2]. It was assumed that crystallisation of the proteins in preparation for x-ray crystallography had left their tertiary structures essentially the same as in solution, but their quaternary (multi-subunit) structures were possibly very different.
This poster will show a new model structure for haemoglobin based on the same concepts. The new structure is consistent with the complex chemical properties of haemoglobin and is supported by cross-linking studies.
Natural haemoglobin occurs as an α2β2 tetramer (α and β being different forms of haemoglobin) that shows cooperative binding of O2. There are also other interactions between the O2 binding, the pH and the concentrations of certain other chemicals. These interactions require that a binding site at one location in the tetramer does work on binding sites elsewhere in the tetramer. Some degree of rigidity would be required to allow specific transmission of this energy to the correct location.
The widely accepted structure of haemoglobin is based on x-ray crystallography, initially by Muirhead and Perutz in 1963. (Some recent NMR studies suggest that the solution structure is similar to the crystal, but they have problems with assumptions and verifiability.) In 1965 Monod, Wyman and Changeux proposed a new mechanism to explain cooperative O2 binding, as it was previously thought that the hemes interacted directly [3]. In Perutz’s structure the hemes could not directly interact and an intricate mechanical and chemical mechanism was proposed. As the complexity of haemoglobin chemistry has emerged since then, much ingenuity has been applied to modifying the models to fit the data. However, serious inconsistencies remain [4, 5]
The new quaternary structure to be shown in this poster differs markedly from Perutz’s structure. It provides an explanation for the apparent necessity that highly cooperative O2 binding involves α2β2 tetramers rather than homotetramers. The “switch” between high and low affinity forms involves isomeric quaternary structures only achievable with an α2β2 tetramer. The interactions between sites are mediated by delocalised π-electrons connected through the rigid central core of the tetramer. This mechanism is reminiscent of the mechanism proposed by Pauling before the x-ray crystal structure became known [3].
[1] Vanselow, D. G. Biophys. J. 2002, 82, 2293-2203.
[2] Vanselow, D. G. Native Proteins; http://nativeproteins.blogspot.com, 2008.
[3] Eaton, W.A.; Henry. E.R.; Hofrichter, J.; Mozzarelli, A. Nat. Struct. Biol. 1999, 6, 351-358.
[4] Yonetani, T.; Park, S.; Tsuneshige, A.; Imai, K.; Kanaori, K. J. Biol. Chem. 2002, 277, 34508-34520.
[5] Eaton, W. A..; Henry, E. R.; Hofrichter, J.; Bettati, S.; Viappiani, C.; Mozzarelli, A. IUBMB Life 2007, 59, 586-599.

A preprint of a full paper describing this work will be posted after the conference.

Proposed tetrahedral quaternary structure for Galactose Oxidase.

Galactose Oxidase has structural similarity to neuraminidase but its function and active site are very different. This preprint shows how a compact tetrahedral structure can be assembled from the X-ray crystal structure. This is the first protein reconstruction that confronts earlier physical measurements that suggest the native form is a monomer. The preprint therefore addresses the limitations of the earlier physical measurements.
Galactose oxidase is a copper enzyme with an aromatic electron-relay system connecting copper ions in adjacent subunits, thus allowing two-electron reduction of molecular oxygen. Apart from this novel feature the enzyme is similar to neuraminidases in having a channel from the active site to the enzyme exterior in order that substrate moieties can be oxidised while still part of a polysaccharide or glycoprotein.

Link here to the preprint.

Feedback most welcome.

Can you help to get this work published and more widely known?

I am interested to hear from anyone with a suggestion about a journal that might be receptive to the neuraminidase work. I would be especially delighted to hear from anyone who would be prepared to act as a referee.
Please bring this BLOG to the attention of any of your colleagues who might be interested.

Expert reviews of neuraminidase paper with author's comments

I have put together the reviews I received on the two occasions I tried to publish this work in high-profile journals. I have included my formal responses and later annotations. The reviews indicate a generally defensive position by X-ray crystallography experts. Have a look. I would appreciate your views.

Link

Please submit a Posting or email me direct at dvanselow@hotmail.com

Don Vanselow

New Native Structures in Neuraminidases

Following on from the theory, I have produced model quaternary structures for a number of neuraminidase enzymes, based on the x-ray crystal structures. Click on these links to view this paper. html linked here. An earlier pdf version is linked here. The paper is copyright 2007 to myself, Dr Don Vanselow, 54 Greenways Road, Glen Waverley, Victoria 3150. Australia. All rights reserved.

The Fundamental Biophysics of Protein Function

The theory of catalysis and other functions displayed by soft biomaterials such as proteins is described in the paper "The Role of Constraint in Catalysis and High-Affinity Binding by Proteins" published in the Biophysical Journal in May 2002. Use the link provided by clicking on the title of this Post.