Dynamic nature of proteins : interpretation of residual dipolar couplings

Protein conformations and dynamics can be studied by nuclear magnetic resonance spectroscopy using dilute liquid crystalline samples. This work clarifies the interpretation of residual dipolar coupling data yielded by the experiments. It was discovered that unfolded proteins without any additional structure beyond that of a mere polypeptide chain exhibit residual dipolar couplings. Also, it was found that molecular dynamics induce fluctuations in the molecular alignment and doing so affect residual dipolar couplings. The finding clarified the origins of low order parameter values observed earlier. The work required the development of new analytical and computational methods for the prediction of intrinsic residual dipolar coupling profiles for unfolded proteins. The presented characteristic chain model is able to reproduce the general trend of experimental residual dipolar couplings for denatured proteins. The details of experimental residual dipolar coupling profiles are beyond the analytical model, but improvements are proposed to achieve greater accuracy. A computational method for rapid prediction of unfolded protein residual dipolar couplings was also developed. Protein dynamics were shown to modulate the effective molecular alignment in a dilute liquid crystalline medium. The effects were investigated from experimental and molecular dynamics generated conformational ensembles of folded proteins. It was noted that dynamics induced alignment is significant especially for the interpretation of molecular dynamics in small, globular proteins. A method of correction was presented. Residual dipolar couplings offer an attractive possibility for the direct observation of protein conformational preferences and dynamics. The presented models and methods of analysis provide significant advances in the interpretation of residual dipolar coupling data from proteins.Proteiinit ovat elämän kannalta välttämättömiä molekyylejä. Solujen toiminta perustuu pitkälti proteiineihin, joita solut valmistavat DNA:ssa olevien "rakennusohjeiden" mukaisesti. Proteiineihin liittyvät häiriöt ovat syypää mm. sellaisiin vakaviin saurauksiin kuin Alzheimerin ja Creutzfeldt-Jakobin taudit. Proteiineilla on yleensä hyvin määritelty ja kohtuullisen vakaa rakenne, mutta usein proteiini sisältää myös heikosti rakenteellisia osia. On olemassa myös rakenteellisesti epävakaita proteiineja, jotka ottavat toiminnallisen muotonsa vasta vuorovaikutuksessa. Kaikki proteiinit ovat kuitenkin jatkuvassa sisäisessä liikkeessä; niiden muoto elää alati. Tässä työssä on tutkittu proteiinien rakenteellisten ja dynaamisten ominaisuuksien tulkintaa ydinmagneettisella resonanssispektroskopialla mitatuista tuloksista. Siinä on osoitettu, että vastoin aiempaa käsitystä myös täysin rakenteettomista proteiineista saadaan mitattua jäännösdipolikytkennäksi kutsuttu suure, joka antaa ainutlaatuista tietoa molekyylin atomien välisistä suhteista. Työssä on myös osoitettu riittämättömäksi vallitseva tapa kohdella proteiinia kuin kiinteää molekyyliä sen keskimääräistä asentoa määritettäessä. Tutkimuksen tulokset mahdollistavat entistä tarkemman kuvan saamisen proteiinien rakenteellisista ja dynaamisista ominaisuuksista. Proteiinien ominaisuuksien lisääntynyt ymmärrys voi mahdollistaa mm. entistä tehokkaampien lääkeaineiden suunnittelun ja kehittämisen

Structural Investigation of MscL Gating Using Experimental Data and Coarse Grained MD Simulations

The mechanosensitive channel of large conductance (MscL) has become a model system in which to understand mechanosensation, a process involved in osmoregulation and many other physiological functions. While a high resolution closed state structure is available, details of the open structure and the gating mechanism remain unknown. In this study we combine coarse grained simulations with restraints from EPR and FRET experiments to study the structural changes involved in gating with much greater level of conformational sampling than has previously been possible. We generated a set of plausible open pore structures that agree well with existing open pore structures and gating models. Most interestingly, we found that membrane thinning induces a kink in the upper part of TM1 that causes an outward motion of the periplasmic loop away from the pore centre. This previously unobserved structural change might present a new mechanism of tension sensing and might be related to a functional role in osmoregulation.The study was supported by a grant from the Australian Research Council. The simulations were carried out using computer time from iVEC and a Merit Allocation Scheme on the NCI National Facility at the Australian National University. ED was supported by a Jean Rogerson Postgraduate scholarship and the Beryl Henderson Memorial Grant by the Australian Federation of University Women ACT. Websites of funding agencies: http://nci.org.au/access/merit-allocationscheme/, http://www.ivec.org/ http://www.arc.gov.au/ncgp/default.htm, http://spe.publishing.uwa.edu.au/latest/scholarships/postgraduate/rogerson, http://www. afgw.org.au/what-we-do/scholarships-2/ The authors hereby confirm that the funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Evidence of molecular alignment fluctuations in aqueous dilute liquid crystalline media

Protein dynamics can be studied by NMR measurements of aqueous dilute liquid crystalline samples. However, the measured residual dipolar couplings are sensitive not only to internal fluctuations but to all changes in internuclear vectors relative to the laboratory frame. We show that side-chain fluctuations and bond librations in the ps-ns time scale perturb the molecular shape and charge distribution of a small globular protein sufficiently to cause a noticeable variation in the molecular alignment. The alignment variation disperses the bond vectors of a conformational ensemble even further from the dispersion already caused by internal fluctuations of a protein. Consequently RDC-probed order parameters are lower than those obtained by laboratory frame relaxation measurements

Vibrational Spectra of a Mechanosensitive Channel

We report the simulated vibrational spectra of a mechanosensitive membrane channel in different gating states. Our results show that while linear absorption is insensitive to structural differences, linear dichroism and sum-frequency generation spectroscopies are sensitive to the orientation of the transmembrane helices, which is changing during the opening process. Linear dichroism cannot distinguish an intermediate structure from the closed structure, but sum-frequency generation can. In addition, we find that two-dimensional infrared spectroscopy can be used to distinguish all three investigated gating states of the mechanosensitive membrane channel.

GPAW: open Python package for electronic-structure calculations

We review the GPAW open-source Python package for electronic structure calculations. GPAW is based on the projector-augmented wave method and can solve the self-consistent density functional theory (DFT) equations using three different wave-function representations, namely real-space grids, plane waves, and numerical atomic orbitals. The three representations are complementary and mutually independent and can be connected by transformations via the real-space grid. This multi-basis feature renders GPAW highly versatile and unique among similar codes. By virtue of its modular structure, the GPAW code constitutes an ideal platform for implementation of new features and methodologies. Moreover, it is well integrated with the Atomic Simulation Environment (ASE) providing a flexible and dynamic user interface. In addition to ground-state DFT calculations, GPAW supports many-body GW band structures, optical excitations from the Bethe-Salpeter Equation (BSE), variational calculations of excited states in molecules and solids via direct optimization, and real-time propagation of the Kohn-Sham equations within time-dependent DFT. A range of more advanced methods to describe magnetic excitations and non-collinear magnetism in solids are also now available. In addition, GPAW can calculate non-linear optical tensors of solids, charged crystal point defects, and much more. Recently, support of GPU acceleration has been achieved with minor modifications of the GPAW code thanks to the CuPy library. We end the review with an outlook describing some future plans for GPAW