RESUMO
In order to become bioactive, proteins must be translated and protected from aggregation during biosynthesis. The ribosome and molecular chaperones play a key role in this process. Ribosome-bound nascent chains (RNCs) of intrinsically disordered proteins and RNCs bearing a signal/arrest sequence are known to interact with ribosomal proteins. However, in the case of RNCs bearing foldable protein sequences, not much information is available on these interactions. Here, via a combination of chemical crosslinking and time-resolved fluorescence-anisotropy, we find that nascent chains of the foldable globin apoHmp1-140 interact with ribosomal protein L23 and have a freely-tumbling non-interacting N-terminal compact region comprising 63-94 residues. Longer RNCs (apoHmp1-189) also interact with an additional yet unidentified ribosomal protein, as well as with chaperones. Surprisingly, the apparent strength of RNC/r-protein interactions does not depend on nascent-chain sequence. Overall, foldable nascent chains establish and expand interactions with selected ribosomal proteins and chaperones, as they get longer. These data are significant because they reveal the interplay between independent conformational sampling and nascent-protein interactions with the ribosomal surface.
Assuntos
Dobramento de Proteína , Proteínas Ribossômicas , Ribossomos , Ribossomos/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/química , Ligação Proteica , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Biossíntese de Proteínas , Modelos Moleculares , Conformação Proteica , HumanosRESUMO
Rocky Mountain spotted fever is caused by Rickettsia rickettsii infection. R. rickettsii can be transmitted to mammals, including humans, through the bite of an infected hard-bodied tick of the family Ixodidae. Since the R. rickettsii genome contains only one cold-shock-like protein and given the essential nature of cold-shock proteins in other bacteria, the structure of the cold-shock-like protein from R. rickettsii was investigated. With the exception of a short α-helix found between ß-strands 3 and 4, the solution structure of the R. rickettsii cold-shock-like protein has the typical Greek-key five-stranded ß-barrel structure found in most cold-shock domains. Additionally, the R. rickettsii cold-shock-like protein, with a ΔG of unfolding of 18.4â kJâ mol(-1), has a similar stability when compared with other bacterial cold-shock proteins.
Assuntos
Proteínas de Bactérias/química , Proteínas e Peptídeos de Choque Frio/química , Rickettsia rickettsii , Sequência de Aminoácidos , Sequência Conservada , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Desdobramento de Proteína , TermodinâmicaRESUMO
The influence of the ribosome on nascent chains is poorly understood, especially in the case of proteins devoid of signal or arrest sequences. Here, we provide explicit evidence for the interaction of specific ribosomal proteins with ribosome-bound nascent chains (RNCs). We target RNCs pertaining to the intrinsically disordered protein PIR and a number of mutants bearing a variable net charge. All the constructs analyzed in this work lack N-terminal signal sequences. By a combination chemical crosslinking and Western-blotting, we find that all RNCs interact with ribosomal protein L23 and that longer nascent chains also weakly interact with L29. The interacting proteins are spatially clustered on a specific region of the large ribosomal subunit, close to the exit tunnel. Based on chain-length-dependence and mutational studies, we find that the interactions with L23 persist despite drastic variations in RNC sequence. Importantly, we also find that the interactions are highly Mg+2-concentration-dependent. This work is significant because it unravels a novel role of the ribosome, which is shown to engage with the nascent protein chain even in the absence of signal or arrest sequences.